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Much of the research in the Pringle laboratory exploits the power of yeast as an experimentally tractable model eukaryote to investigate fundamental problems in cell and developmental biology such as the mechanisms of cell polarization and cytokinesis. In regards to cell polarization, the major current foci are the roles of cortical marker proteins and of a GTPase-based signal-transduction cascade in the selection of the polarization axes (as defined by the bud sites). Interestingly, the marker proteins appear to be delivered to polarized sites in the cell surface by an unconventional arm of the secretory pathway. In regards to cytokinesis, the major current foci are the roles of the septin proteins and the interactions among the actomyosin contractile ring, the enzymes of extracellular-matrix (cell-wall) synthesis, and proteins that appear to be involved in plasma-membrane reorganization. Our working hypothesis is that the conserved core mechanism is the rearrangements of the membrane during cleavage-furrow formation and that the actomyosin ring and extracellular matrix play accessory roles. In a departure from our many years of yeast work, a major new project involves developing the small sea anemone Aiptasia pallida as a model system for study of the molecular and cellular biology of the dinoflagellate-cnidarian symbiosis, which is critical for the survival of most reef-building corals but still very poorly understood. Processes to be investigated include the recognition and signaling events involved in symbiosis establishment, the temporal and spatial coordination of symbiont and host cell cycles during symbiosis maintenance, and the signaling and cellular processes involved in symbiosis breakdown under stress. Currently much of our effort is directed at genomic analysis and method development that will underpin later studies.

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Abstract

The unicellular green alga Chlamydomonas reinhardtii is a model organism that provides an opportunity to understand the evolution and functional biology of the lineage that includes the land plants, as well as aspects of the fundamental core biology conserved throughout the eukaryotic phylogeny. Although many tools are available to facilitate genetic, molecular biological, biochemical, and cell biological studies in Chlamydomonas, expression of unselected transgenes of interest (GOIs) has been challenging. In most methods used previously, the GOI and a selectable marker are expressed from two separate mRNAs, so that their concomitant expression is not guaranteed. In this study, we developed constructs that allow expression of an upstream GOI and downstream selectable marker from a single bicistronic mRNA. Although this approach in other systems has typically required a translation-enhancing element such as an internal ribosome entry site for the downstream marker, we found that a short stretch of unstructured junction sequence was sufficient to obtain adequate expression of the downstream gene, presumably through post-termination reinitiation. With this system, we obtained robust expression of both endogenous and heterologous GOIs, including fluorescent proteins and tagged fusion proteins, in the vast majority of transformants, thus eliminating the need for tedious secondary screening for GOI-expressing transformants. This improved efficiency should greatly facilitate a variety of genetic and cell-biological studies in Chlamydomonas and also enable new applications such as expression-based screens and large-scale production of foreign proteins.

Abstract

When exposed to stress such as high seawater temperature, corals and other cnidarians can bleach due to loss of symbiotic algae from the host tissue and/or loss of pigments from the algae. Although the environmental conditions that trigger bleaching are reasonably well known, its cellular and molecular mechanisms are not well understood. Previous studies have reported the occurrence of at least four different cellular mechanisms for the loss of symbiotic algae from the host tissue: in situ degradation of algae, exocytic release of algae from the host, detachment of host cells containing algae, and death of host cells containing algae. The relative contributions of these several mechanisms to bleaching remain unclear, and it is also not known whether these relative contributions change in animals subjected to different types and/or durations of stresses. In this study, we used a clonal population of the small sea anemone Aiptasia, exposed individuals to various precisely controlled stress conditions, and quantitatively assessed the several possible bleaching mechanisms in parallel. Under all stress conditions tested, except for acute cold shock at 4°C, expulsion of intact algae from the host cells appeared to be by far the predominant mechanism of bleaching. During acute cold shock, in situ degradation of algae and host-cell detachment also became quantitatively significant, and the algae released under these conditions appeared to be severely damaged.

Abstract

Actin is one of the most conserved eukaryotic proteins. It is thought to have multiple essential cellular roles and to function primarily or exclusively as filaments ("F-actin"). Chlamydomonas has been an enigma, because a null mutation (ida5-1) in its single gene for conventional actin does not affect growth. A highly divergent actin gene, NAP1, is upregulated in ida5-1 cells, but it has been unclear whether NAP1 can form filaments or provide actin function. Here, we used the actin-depolymerizing drug latrunculin B (LatB), the F-actin-specific probe Lifeact-Venus, and genetic and molecular methods to resolve these issues. LatB-treated wild-type cells continue to proliferate; they initially lose Lifeact-stained structures but recover them concomitant with upregulation of NAP1. Thirty-nine LatB-sensitive mutants fell into four genes (NAP1 and LAT1-LAT3) in which we identified the causative mutations using a novel combinatorial pool-sequencing strategy. LAT1-LAT3 are required for NAP1 upregulation upon LatB treatment, and ectopic expression of NAP1 largely rescues the LatB sensitivity of the lat1-lat3 mutants, suggesting that the LAT gene products comprise a regulatory hierarchy with NAP1 expression as the major functional output. Selection of LatB-resistant revertants of a nap1 mutant yielded dominant IDA5 mutations that presumably render F-IDA5 resistant to LatB, and nap1 and lat mutations are synthetically lethal with ida5-1 in the absence of LatB. We conclude that both IDA5 and the divergent NAP1 can form filaments and redundantly provide essential F-actin functions and that a novel surveillance system, probably responding to a loss of F-actin, triggers NAP1 expression and perhaps other compensatory responses.

Abstract

Rho-type small GTPases are involved in cytokinesis in various organisms, but their precise roles and regulation remain unclear. Rho proteins function as molecular switches by cycling between the active GTP-bound and inactive GDP-bound states; the GTP-bound proteins in turn interact with their downstream effectors to transmit the signal. Biochemical assays using Rho-binding domains of effector proteins have been used to specifically pull down GTP-bound Rho proteins from cell extracts. Here, we describe the application of such a method in combination with cell-cycle synchronization in the budding yeast Saccharomyces cerevisiae; this approach allows dissection of the activity of Rho1 at different stages of cytokinesis. We also present data showing the importance of caution in interpreting such biochemical data and of comparing to the results obtained with other approaches where possible. The principle of this protocol is also applicable to analyses of other Rho-type GTPases and cell-cycle events.

The nonopisthokont septins: How many there are, how little we know about them, and how we might learn more.Methods in cell biologyOnishi, M., Pringle, J. R.2016; 136: 1-19

Abstract

We have confirmed and extended previous reports of a wide distribution of septin proteins in the eukaryotic phylogeny. It now appears that septins are present in at least some representatives of every eukaryotic supergroup, with the possible exception of the Excavata. Presently, almost nothing is known of the structure, assembly, and biological roles of septins outside of the opisthokonts (animals, fungi, and their close relatives). Thus, studies of the septins in the highly diverse and distantly related nonopisthokont groups present a major opportunity to gain a much deeper understanding of septin core function and evolution, and we discuss briefly the excellent prospects for capitalizing on this opportunity in the next few years.

Abstract

Dicentric chromosomes are unstable products of erroneous DNA repair events that can lead to further genome rearrangements and extended gene copy number variations. During mitosis, they form anaphase bridges, resulting in chromosome breakage by an unknown mechanism. In budding yeast, dicentrics generated by telomere fusion break at the fusion, a process that restores the parental karyotype and protects cells from rare accidental telomere fusion. Here, we observed that dicentrics lacking telomere fusion preferentially break within a 25- to 30-kb-long region next to the centromeres. In all cases, dicentric breakage requires anaphase exit, ruling out stretching by the elongated mitotic spindle as the cause of breakage. Instead, breakage requires cytokinesis. In the presence of dicentrics, the cytokinetic septa pinch the nucleus, suggesting that dicentrics are severed after actomyosin ring contraction. At this time, centromeres and spindle pole bodies relocate to the bud neck, explaining how cytokinesis can sever dicentrics near centromeres.

Abstract

In studies of both the establishment and breakdown of cnidarian-dinoflagellate symbiosis, it is often necessary to determine the number of Symbiodinium cells relative to the quantity of host tissue. Ideally, the methods used should be rapid, precise, and accurate. In this study, we systematically evaluated methods for sample preparation and storage and the counting of algal cells using the hemocytometer, a custom image-analysis program for automated counting of the fluorescent algal cells, the Coulter Counter, or the Millipore Guava flow-cytometer. We found that although other methods may have value in particular applications, for most purposes, the Guava flow cytometer provided by far the best combination of precision, accuracy, and efficient use of investigator time (due to the instrument's automated sample handling), while also allowing counts of algal numbers over a wide range and in small volumes of tissue homogenate. We also found that either of two assays of total homogenate protein provided a precise and seemingly accurate basis for normalization of algal counts to the total amount of holobiont tissue.

Abstract

Centrosomes play critical roles in the cell division cycle and ciliogenesis. Sfi1 is a centrin-binding protein conserved from yeast to humans. Budding yeast Sfi1 is essential for the initiation of spindle pole body (SPB; yeast centrosome) duplication. However, the recruitment and partitioning of Sfi1 to centrosomal structures have never been fully investigated in any organism, and the presumed importance of the conserved tryptophans in the internal repeats of Sfi1 remains untested. Here we report that in fission yeast, instead of doubling abruptly at the initiation of SPB duplication and remaining at a constant level thereafter, Sfi1 is gradually recruited to SPBs throughout the cell cycle. Like an sfi1Δ mutant, a Trp-to-Arg mutant (sfi1-M46) forms monopolar spindles and exhibits mitosis and cytokinesis defects. Sfi1-M46 protein associates preferentially with one of the two daughter SPBs during mitosis, resulting in a failure of new SPB assembly in the SPB receiving insufficient Sfi1. Although all five conserved tryptophans tested are involved in Sfi1 partitioning, the importance of the individual repeats in Sfi1 differs. In summary, our results reveal a link between the conserved tryptophans and Sfi1 partitioning and suggest a revision of the model for SPB assembly.

Similar specificities of symbiont uptake by adults and larvae in an anemone model system for coral biologyJOURNAL OF EXPERIMENTAL BIOLOGYHambleton, E. A., Guse, A., Pringle, J. R.2014; 217 (9): 1613-1619

Abstract

Reef-building corals depend for much of their energy on photosynthesis by symbiotic dinoflagellate algae (genus Symbiodinium) that live within their gastrodermal cells. However, the cellular mechanisms underpinning this ecologically critical symbiosis, including those governing the specificity of symbiont uptake by the host, remain poorly understood, in part because of the difficulties of working with corals in the laboratory. Here, we used the small symbiotic sea anemone Aiptasia as an experimentally tractable model system to analyze the specificity and timing of symbiosis onset in larval and adult animals under controlled laboratory conditions. Using four clonal, axenic Symbiodinium strains, we found no difference in uptake specificity between larvae (even when very young) and adults. Although both compatible and incompatible algal strains were found within the larval guts, only the former appeared to be internalized by gastrodermal cells, and they (but not incompatible algae) proliferated rapidly within the larvae in the absence of detectable exchange with other larvae. Older larvae showed reduced ingestion of both compatible and incompatible algae, and the addition of food failed to promote the uptake of an incompatible algal strain. Thus, Aiptasia adults and larvae appear to have similar mechanisms for discriminating between compatible and incompatible dinoflagellate types prior to phagocytosis by host gastrodermal cells. Whether a particular algal strain is compatible or incompatible appears to be stable during years of axenic culture in the absence of a host. These studies provide a foundation for future analyses of the mechanisms of symbiont-uptake specificity in this emerging model system.

Abstract

Coral reefs provide habitats for a disproportionate number of marine species relative to the small area of the oceans that they occupy. The mutualism between the cnidarian animal hosts and their intracellular dinoflagellate symbionts provides the nutritional foundation for coral growth and formation of reef structures, because algal photosynthesis can provide >90% of the total energy of the host. Disruption of this symbiosis ("coral bleaching") is occurring on a large scale due primarily to anthropogenic factors and poses a major threat to the future of coral reefs. Despite the importance of this symbiosis, the cellular mechanisms involved in its establishment, maintenance, and breakdown remain largely unknown. We report our continued development of genomic tools to study these mechanisms in Aiptasia, a small sea anemone with great promise as a model system for studies of cnidarian-dinoflagellate symbiosis. Specifically, we have generated de novo assemblies of the transcriptomes of both a clonal line of symbiotic anemones and their endogenous dinoflagellate symbionts. We then compared transcript abundances in animals with and without dinoflagellates. This analysis identified >900 differentially expressed genes and allowed us to generate testable hypotheses about the cellular functions affected by symbiosis establishment. The differentially regulated transcripts include >60 encoding proteins that may play roles in transporting various nutrients between the symbiotic partners; many more encoding proteins functioning in several metabolic pathways, providing clues regarding how the transported nutrients may be used by the partners; and several encoding proteins that may be involved in host recognition and tolerance of the dinoflagellate.

Abstract

The global decline of reef-building corals is due in part to the loss of algal symbionts, or "bleaching," during the increasingly frequent periods of high seawater temperatures [1, 2]. During bleaching, endosymbiotic dinoflagellate algae (Symbiodinium spp.) either are lost from the animal tissue or lose their photosynthetic pigments, resulting in host mortality if the Symbiodinium populations fail to recover [3]. The >1,000 studies of the causes of heat-induced bleaching have focused overwhelmingly on the consequences of damage to algal photosynthetic processes [4-6], and the prevailing model for bleaching invokes a light-dependent generation of toxic reactive oxygen species (ROS) by heat-damaged chloroplasts as the primary trigger [6-8]. However, the precise mechanisms of bleaching remain unknown, and there is evidence for involvement of multiple cellular processes [9, 10]. In this study, we asked the simple question of whether bleaching can be triggered by heat in the dark, in the absence of photosynthetically derived ROS. We used both the sea anemone model system Aiptasia [11, 12] and several species of reef-building corals to demonstrate that symbiont loss can occur rapidly during heat stress in complete darkness. Furthermore, we observed damage to the photosynthetic apparatus under these conditions in both Aiptasia endosymbionts and cultured Symbiodinium. These results do not directly contradict the view that light-stimulated ROS production is important in bleaching, but they do show that there must be another pathway leading to bleaching. Elucidation of this pathway should help to clarify bleaching mechanisms under the more usual conditions of heat stress in the light.

Abstract

In yeast and animal cytokinesis, the small guanosine triphosphatase (GTPase) Rho1/RhoA has an established role in formation of the contractile actomyosin ring, but its role, if any, during cleavage-furrow ingression and abscission is poorly understood. Through genetic screens in yeast, we found that either activation of Rho1 or inactivation of another small GTPase, Cdc42, promoted secondary septum (SS) formation, which appeared to be responsible for abscission. Consistent with this hypothesis, a dominant-negative Rho1 inhibited SS formation but not cleavage-furrow ingression or the concomitant actomyosin ring constriction. Moreover, Rho1 is temporarily inactivated during cleavage-furrow ingression; this inactivation requires the protein Cyk3, which binds Rho1-guanosine diphosphate via its catalytically inactive transglutaminase-like domain. Thus, unlike the active transglutaminases that activate RhoA, the multidomain protein Cyk3 appears to inhibit activation of Rho1 (and thus SS formation), while simultaneously promoting cleavage-furrow ingression through primary septum formation. This work suggests a general role for the catalytically inactive transglutaminases of fungi and animals, some of which have previously been implicated in cytokinesis.

Role of endocytosis in localization and maintenance of the spatial markers for bud-site selection in yeast.PloS oneTuo, S., Nakashima, K., Pringle, J. R.2013; 8 (9)

Abstract

The yeast Saccharomyces cerevisiae normally selects bud sites (and hence axes of cell polarization) in one of two distinct patterns, the axial pattern of haploid cells and the bipolar pattern of diploid cells. These patterns depend on distinct sets of cortical-marker proteins that transmit positional information through a common signaling pathway based on a Ras-type GTPase. It has been reported previously that various proteins of the endocytic pathway may be involved in determining the bipolar pattern but not the axial pattern. To explore this question systematically, we constructed and analyzed congenic haploid and diploid deletion mutants for 14 genes encoding proteins that are involved in endocytosis. The mutants displayed a wide range of severities in their overall endocytosis defects, as judged by their growth rates and abilities to take up the lipophilic dye FM 4-64. Consistent with the previous reports, none of the mutants displayed a significant defect in axial budding, but they displayed defects in bipolar budding that were roughly correlated with the severities of their overall endocytosis defects. Both the details of the mutant budding patterns and direct examination of GFP-tagged marker proteins suggested that both initial formation and maintenance of the normally persistent bipolar marks depend on endocytosis, as well as polarized exocytosis, in actively growing cells. Interestingly, maintenance of the bipolar marks in non-growing cells did not appear to require normal levels of endocytosis. In some cases, there was a striking lack of correlation between the overall severities of the general-endocytosis defect and the bud-site selection defect, suggesting that various endocytosis proteins may differ in their importance for the uptake of various plasma-membrane targets.

Apparent Defect in Yeast Bud-Site Selection Due to a Specific Failure to Splice the Pre-mRNA of a Regulator of Cell-Type-Specific TranscriptionPLOS ONETuo, S., Nakashima, K., Pringle, J. R.2012; 7 (10)

Abstract

The yeast Saccharomyces cerevisiae normally selects bud sites (and hence axes of cell polarization) in one of two distinct patterns, the axial pattern of haploid cells and the bipolar pattern of diploid cells. Although many of the proteins involved in bud-site selection are known, it is likely that others remain to be identified. Confirming a previous report (Ni and Snyder, 2001, Mol. Biol. Cell 12, 2147-2170), we found that diploids homozygous for deletions of IST3/SNU17 or BUD13 do not show normal bipolar budding. However, these abnormalities do not reflect defects in the apparatus of bipolar budding. Instead, the absence of Ist3 or Bud13 results in a specific defect in the splicing of the MATa1 pre-mRNA, which encodes a repressor that normally blocks expression of haploid-specific genes in diploid cells. When Mata1 protein is lacking, Axl1, a haploid-specific protein critical for the choice between axial and bipolar budding, is expressed ectopically in diploid cells and disrupts bipolar budding. The involvement of Ist3 and Bud13 in pre-mRNA splicing is by now well known, but the degree of specificity shown here for MATa1 pre-mRNA, which has no obvious basis in the pre-mRNA structure, is rather surprising in view of current models for the functions of these proteins. Moreover, we found that deletion of PML1, whose product is thought to function together with Ist3 and Bud13 in a three-protein retention-and-splicing (RES) complex, had no detectable effect on the splicing in vivo of either MATa1 or four other pre-mRNAs.

Abstract

Reef-building corals and many other cnidarians are symbiotic with dinoflagellates of the genus Symbiodinium. It has long been known that the endosymbiotic algae transfer much of their photosynthetically fixed carbon to the host and that this can provide much of the host's total energy. However, it has remained unclear which metabolite(s) are directly translocated from the algae into the host tissue. We reexamined this question in the small sea anemone Aiptasia using labeling of intact animals in the light with (13)C-bicarbonate, rapid homogenization and separation of animal and algal fractions, and analysis of metabolite labeling by gas chromatography-mass spectrometry. We found labeled glucose in the animal fraction within 2 min of exposure to (13)C-bicarbonate, whereas no significant labeling of other compounds was observed within the first 10 min. Although considerable previous evidence has suggested that glycerol might be a major translocated metabolite, we saw no significant labeling of glycerol within the first hour, and incubation of intact animals with (13)C-labeled glycerol did not result in a rapid production of (13)C-glucose. In contrast, when Symbiodinium cells freshly isolated from host tissue were exposed to light and (13)C-bicarbonate in the presence of host homogenate, labeled glycerol, but not glucose, was detected in the medium. We also observed early production of labeled glucose, but not glycerol, in three coral species. Taken together, the results suggest that glucose is the major translocated metabolite in dinoflagellate-cnidarian symbiosis and that the release of glycerol from isolated algae may be part of a stress response.

Abstract

Cell morphogenesis is a complex process that relies on a diverse array of proteins and pathways. We have identified a transglutaminase-like protein (Cyk3p) that functions in fission yeast morphogenesis. The phenotype of a cyk3 knockout strain indicates a primary role for Cyk3p in cytokinesis. Correspondingly, Cyk3p localizes both to the actomyosin contractile ring and the division septum, promoting ring constriction, septation, and subsequent cell separation following ring disassembly. In addition, Cyk3p localizes to polarized growth sites and plays a role in cell shape determination, and it also appears to contribute to cell integrity during stationary phase, given its accumulation as dynamic puncta at the cortex of such cells. Our results and the conservation of Cyk3p across fungi point to a role in cell wall synthesis and remodeling. Cyk3p possesses a transglutaminase domain that is essential for function, even though it lacks the catalytic active site. In a wider sense, our work illustrates the physiological importance of inactive members of the transglutaminase family, which are found throughout eukaryotes. We suggest that the proposed evolution of animal transglutaminase cross-linking activity from ancestral bacterial thiol proteases was accompanied by the emergence of a subclass whose function does not depend on enzymatic activity.

Abstract

Coral reefs are hotspots of oceanic biodiversity, forming the foundation of ecosystems that are important both ecologically and for their direct practical impacts on humans. Corals are declining globally due to a number of stressors, including rising sea-surface temperatures and pollution; such stresses can lead to a breakdown of the essential symbiotic relationship between the coral host and its endosymbiotic dinoflagellates, a process known as coral bleaching. Although the environmental stresses causing this breakdown are largely known, the cellular mechanisms of symbiosis establishment, maintenance, and breakdown are still largely obscure. Investigating the symbiosis using an experimentally tractable model organism, such as the small sea anemone Aiptasia, should improve our understanding of exactly how the environmental stressors affect coral survival and growth.We assembled the transcriptome of a clonal population of adult, aposymbiotic (dinoflagellate-free) Aiptasia pallida from ~208 million reads, yielding 58,018 contigs. We demonstrated that many of these contigs represent full-length or near-full-length transcripts that encode proteins similar to those from a diverse array of pathways in other organisms, including various metabolic enzymes, cytoskeletal proteins, and neuropeptide precursors. The contigs were annotated by sequence similarity, assigned GO terms, and scanned for conserved protein domains. We analyzed the frequency and types of single-nucleotide variants and estimated the size of the Aiptasia genome to be ~421 Mb. The contigs and annotations are available through NCBI (Transcription Shotgun Assembly database, accession numbers JV077153-JV134524) and at http://pringlelab.stanford.edu/projects.html.The availability of an extensive transcriptome assembly for A. pallida will facilitate analyses of gene-expression changes, identification of proteins of interest, and other studies in this important emerging model system.

Abstract

Ultra-high-throughput sequencing produces duplicate and near-duplicate reads, which can consume computational resources in downstream applications. A tool that collapses such reads should reduce storage and assembly complications and costs.We developed Fulcrum to collapse identical and near-identical Illumina and 454 reads (such as those from PCR clones) into single error-corrected sequences; it can process paired-end as well as single-end reads. Fulcrum is customizable and can be deployed on a single machine, a local network or a commercially available MapReduce cluster, and it has been optimized to maximize ease-of-use, cross-platform compatibility and future scalability. Sequence datasets have been collapsed by up to 71%, and the reduced number and improved quality of the resulting sequences allow assemblers to produce longer contigs while using less memory.

Abstract

Septins are essential for cytokinesis in Saccharomyces cerevisiae, but their precise roles remain elusive. Currently, it is thought that before cytokinesis, the hourglass-shaped septin structure at the mother-bud neck acts as a scaffold for assembly of the actomyosin ring (AMR) and other cytokinesis factors. At the onset of cytokinesis, the septin hourglass splits to form a double ring that sandwiches the AMR and may function as diffusion barriers to restrict diffusible cytokinesis factors to the division site. Here, we show that in cells lacking the septin Cdc10 or the septin-associated protein Bud4, the septins form a ring-like structure at the mother-bud neck that fails to re-arrange into a double ring early in cytokinesis. Strikingly, AMR assembly and constriction, the localization of membrane-trafficking and extracellular-matrix-remodeling factors, cytokinesis, and cell-wall-septum formation all occur efficiently in cdc10Δ and bud4Δ mutants. Thus, diffusion barriers formed by the septin double ring do not appear to be critical for S. cerevisiae cytokinesis. However, an AMR mutation and a septin mutation have synergistic effects on cytokinesis and the localization of cytokinesis proteins, suggesting that tethering to the AMR and a septin diffusion barrier may function redundantly to localize proteins to the division site.

Abstract

Until recently, it had appeared that the septin family of proteins was restricted to the opisthokont eukaryotes (the fungi and animals and their close relatives the microsporidia and choanoflagellates). It has now become apparent that septins are also present in several other widely divergent eukaryotic lineages (chlorophyte algae, brown algae, and ciliates). This distribution and the details of the non-opisthokont septin sequences appear to require major revisions to hypotheses about the origins and early evolution of the septins.

Abstract

A major question about cytokinesis concerns the role of the septin proteins, which localize to the division site in all animal and fungal cells but are essential for cytokinesis only in some cell types. For example, in Schizosaccharomyces pombe, four septins localize to the division site, but deletion of the four genes produces only a modest delay in cell separation. To ask if the S. pombe septins function redundantly in cytokinesis, we conducted a synthetic-lethal screen in a septin-deficient strain and identified seven mutations. One mutation affects Cdc4, a myosin light chain that is an essential component of the cytokinetic actomyosin ring. Five others cause frequent cell lysis during cell separation and map to two loci. These mutations and their dosage suppressors define a signaling pathway (including Rho1 and a novel arrestin) for repairing cell-wall damage. The seventh mutation affects the poorly understood RNA-binding protein Scw1 and severely delays cell separation when combined either with a septin mutation or with a mutation affecting the septin-interacting, anillin-like protein Mid2, suggesting that Scw1 functions in a pathway parallel to that of the septins. Taken together, our results suggest that the S. pombe septins participate redundantly in one or more pathways that cooperate with the actomyosin ring during cytokinesis and that a septin defect causes septum defects that can be repaired effectively only when the cell-integrity pathway is intact.

Abstract

During yeast sporulation, a forespore membrane (FSM) initiates at each spindle-pole body and extends to form the spore envelope. We used Schizosaccharomyces pombe to investigate the role of septins during this process. During the prior conjugation of haploid cells, the four vegetatively expressed septins (Spn1, Spn2, Spn3, and Spn4) coassemble at the fusion site and are necessary for its normal morphogenesis. Sporulation involves a different set of four septins (Spn2, Spn5, Spn6, and the atypical Spn7) that does not include the core subunits of the vegetative septin complex. The four sporulation septins form a complex in vitro and colocalize interdependently to a ring-shaped structure along each FSM, and septin mutations result in disoriented FSM extension. The septins and the leading-edge proteins appear to function in parallel to orient FSM extension. Spn2 and Spn7 bind to phosphatidylinositol 4-phosphate [PtdIns(4)P] in vitro, and PtdIns(4)P is enriched in the FSMs, suggesting that septins bind to the FSMs via this lipid. Cells expressing a mutant Spn2 protein unable to bind PtdIns(4)P still form extended septin structures, but these structures fail to associate with the FSMs, which are frequently disoriented. Thus, septins appear to form a scaffold that helps to guide the oriented extension of the FSM.

Abstract

Cytokinesis requires coordination of actomyosin ring (AMR) contraction with rearrangements of the plasma membrane and extracellular matrix. In Saccharomyces cerevisiae, new membrane, the chitin synthase Chs2 (which forms the primary septum [PS]), and the protein Inn1 are all delivered to the division site upon mitotic exit even when the AMR is absent. Inn1 is essential for PS formation but not for Chs2 localization. The Inn1 C-terminal region is necessary for localization, and distinct PXXP motifs in this region mediate functionally important interactions with SH3 domains in the cytokinesis proteins Hof1 (an F-BAR protein) and Cyk3 (whose overexpression can restore PS formation in inn1Delta cells). The Inn1 N terminus resembles C2 domains but does not appear to bind phospholipids; nonetheless, when overexpressed or fused to Hof1, it can provide Inn1 function even in the absence of the AMR. Thus, Inn1 and Cyk3 appear to cooperate in activating Chs2 for PS formation, which allows coordination of AMR contraction with ingression of the cleavage furrow.

Abstract

The most diverse marine ecosystems, coral reefs, depend upon a functional symbiosis between cnidarian hosts and unicellular dinoflagellate algae. The molecular mechanisms underlying the establishment, maintenance, and breakdown of the symbiotic partnership are, however, not well understood. Efforts to dissect these questions have been slow, as corals are notoriously difficult to work with. In order to expedite this field of research, we generated and analyzed a collection of expressed sequence tags (ESTs) from the sea anemone Aiptasia pallida and its dinoflagellate symbiont (Symbiodinium sp.), a system that is gaining popularity as a model to study cellular, molecular, and genomic questions related to cnidarian-dinoflagellate symbioses.A set of 4,925 unique sequences (UniSeqs) comprising 1,427 clusters of 2 or more ESTs (contigs) and 3,498 unclustered ESTs (singletons) was generated by analyzing 10,285 high-quality ESTs from a mixed host/symbiont cDNA library. Using a BLAST-based approach to predict which unique sequences derived from the host versus symbiont genomes, we found that the contribution of the symbiont genome to the transcriptome was surprisingly small (1.6-6.4%). This may reflect low levels of gene expression in the symbionts, low coverage of alveolate genes in the sequence databases, a small number of symbiont cells relative to the total cellular content of the anemones, or failure to adequately lyse symbiont cells. Furthermore, we were able to identify groups of genes that are known or likely to play a role in cnidarian-dinoflagellate symbioses, including oxidative stress pathways that emerged as a prominent biological feature of this transcriptome. All ESTs and UniSeqs along with annotation results and other tools have been made accessible through the implementation of a publicly accessible database named AiptasiaBase.We have established the first large-scale transcriptomic resource for Aiptasia pallida and its dinoflagellate symbiont. These data provide researchers with tools to study questions related to cnidarian-dinoflagellate symbioses on a molecular, cellular, and genomic level. This groundwork represents a crucial step towards the establishment of a tractable model system that can be utilized to better understand cnidarian-dinoflagellate symbioses. With the advent of next-generation sequencing methods, the transcriptomic inventory of A. pallida and its symbiont, and thus the extent of AiptasiaBase, should expand dramatically in the near future.

Abstract

The anaphase-promoting complex (APC) is a ubiquitin ligase that controls progression through mitosis by targeting specific proteins for degradation. It is unclear whether the APC also contributes to the control of cytokinesis, the process that divides the cell after mitosis. We addressed this question in the yeast Saccharomyces cerevisiae by studying the effects of APC mutations on the actomyosin ring, a structure containing actin, myosin, and several other proteins that forms at the division site and is important for cytokinesis. In wild-type cells, actomyosin-ring constituents are removed progressively from the ring during contraction and disassembled completely thereafter. In cells lacking the APC activator Cdh1, the actomyosin ring contracts at a normal rate, but ring constituents are not disassembled normally during or after contraction. After cytokinesis in mutant cells, aggregates of ring proteins remain at the division site and at additional foci in other parts of the cell. A key target of APC(Cdh1) is the ring component Iqg1, the destruction of which contributes to actomyosin-ring disassembly. Deletion of CDH1 also exacerbates actomyosin-ring disassembly defects in cells with mutations in the myosin light-chain Mlc2, suggesting that Mlc2 and the APC employ independent mechanisms to promote ring disassembly during cytokinesis.

Abstract

Cell shape and membrane remodeling rely on regulated interactions between the lipid bilayer and cytoskeletal arrays at the cell cortex. During cytokinesis, animal cells build an actomyosin ring anchored to the plasma membrane at the equatorial cortex. Ring constriction coupled to plasma-membrane ingression separates the two daughter cells. Plasma-membrane lipids influence membrane biophysical properties such as membrane curvature and elasticity and play an active role in cell function, and specialized membrane domains are emerging as important factors in regulating assembly and rearrangement of the cytoskeleton. Here, we show that mutations in the gene bond, which encodes a Drosophila member of the family of Elovl proteins that mediate elongation of very-long-chain fatty acids, block or dramatically slow cleavage-furrow ingression during early telophase in dividing spermatocytes. In bond mutant cells at late stages of division, the contractile ring frequently detaches from the cortex and constricts or collapses to one side of the cell, and the cleavage furrow regresses. Our findings implicate very-long-chain fatty acids or their derivative complex lipids in allowing supple membrane deformation and the stable connection of cortical contractile components to the plasma membrane during cell division.

Abstract

Corals provide the foundation of important tropical reef ecosystems but are in global decline for multiple reasons, including climate change. Coral health depends on a fragile partnership with intracellular dinoflagellate symbionts. We argue here that progress in understanding coral biology requires intensive study of the cellular processes underlying this symbiosis. Such study will inform us on how the coral symbiosis will be affected by climate change, mechanisms driving coral bleaching and disease, and the coevolution of this symbiosis in the context of other host-microbe interactions. Drawing lessons from the broader history of molecular and cell biology and the study of other host-microbe interactions, we argue that a model-systems approach is essential for making effective progress in understanding coral cell biology.

Abstract

In a URA3/5-FOA-based dosage-suppressor screen, we isolated a plasmid containing the little-characterized ORF YJL055W. Further analysis showed that this gene did not suppress the mutation of interest. Instead, overexpression of Yjl055Wp directly suppressed the non-viability of URA3(+) cells in the presence of 5-FOA. Overexpression of Yjl055Wp also suppressed the lethality induced by 5-FU, but deletion of YJL055W had no detectable effect on resistance to either 5-FOA or 5-FU. Based on these observations and a previous report that a yjl055wDelta mutant has increased sensitivity to purine-analogue mutagens, we suggest that Yjl055Wp may function in one of several pathways for the detoxification of base analogues. However, its precise mechanism of action remains unknown.

Abstract

In the yeast Saccharomyces cerevisiae, a ring of myosin II forms in a septin-dependent manner at the budding site in late G1. This ring remains at the bud neck until the onset of cytokinesis, when actin is recruited to it. The actomyosin ring then contracts, septum formation occurs concurrently, and cytokinesis is soon completed. Deletion of MYO1 (the only myosin II gene) is lethal on rich medium in the W303 strain background and causes slow-growth and delayed-cell-separation phenotypes in the S288C strain background. These phenotypes can be suppressed by deletions of genes encoding nonessential components of the anaphase-promoting complex (APC/C). This suppression does not seem to result simply from a delay in mitotic exit, because overexpression of a nondegradable mitotic cyclin does not suppress the same phenotypes. Overexpression of either IQG1 or CYK3 also suppresses the myo1Delta phenotypes, and Iqg1p (an IQGAP protein) is increased in abundance and abnormally persistent after cytokinesis in APC/C mutants. In vitro assays showed that Iqg1p is ubiquitinated directly by APC/C(Cdh1) via a novel recognition sequence. A nondegradable Iqg1p (lacking this recognition sequence) can suppress the myo1Delta phenotypes even when expressed at relatively low levels. Together, the data suggest that compromise of APC/C function allows the accumulation of Iqg1p, which then promotes actomyosin-ring-independent cytokinesis at least in part by activation of Cyk3p.

Abstract

The yeast cell wall is an essential organelle that protects the cell from mechanical damage and antimicrobial peptides, participates in cell recognition and adhesion, and is important for the generation and maintenance of normal cell shape. We studied the localization of three covalently bound cell wall proteins in Saccharomyces cerevisiae. Tip1p was found only in mother cells, whereas Cwp2p was incorporated in small-to-medium-sized buds. When the promoter regions of TIP1 and CWP2 (responsible for transcription in early G1 and S/G2 phases, respectively) were exchanged, the localization patterns of Tip1p and Cwp2p were reversed, indicating that the localization of cell wall proteins can be completely determined by the timing of transcription during the cell cycle. The third protein, Cwp1p, was incorporated into the birth scar, where it remained for several generations. However, we could not detect any role of Cwp1p in strengthening the birth scar wall or any functional interaction with the proteins that mark the birth scar pole as a potential future budding site. Promoter-exchange experiments showed that expression in S/G2 phase is necessary but not sufficient for the normal localization of Cwp1p. Studies of mutants in which septum formation is perturbed indicate that the normal asymmetric localization of Cwp1p also depends on the normal timing of septum formation, composition of the septum, or both.

Abstract

The septins are GTP-binding, filament-forming proteins that are involved in cytokinesis and other processes. In the yeast Saccharomyces cerevisiae, the septins are recruited to the presumptive bud site at the cell cortex, where they form a ring through which the bud emerges. We report here that in wild-type cells, the septins typically become detectable in the vicinity of the bud site several minutes before ring formation, but the ring itself is the first distinct structure that forms. Septin recruitment depends on activated Cdc42p but not on the normal pathway for bud-site selection. Recruitment occurs in the absence of F-actin, but ring formation is delayed. Mutant phenotypes and suppression data suggest that the Cdc42p effectors Gic1p and Gic2p, previously implicated in polarization of the actin cytoskeleton, also function in septin recruitment. Two-hybrid, in vitro protein binding, and coimmunoprecipitation data indicate that this role involves a direct interaction of the Gic proteins with the septin Cdc12p.

Abstract

In the budding yeast Saccharomyces cerevisiae, selection of the bud site determines the axis of polarized cell growth and eventual oriented cell division. Bud sites are selected in specific patterns depending on cell type. These patterns appear to depend on distinct types of marker proteins in the cell cortex; in particular, the bipolar budding of diploid cells depends on persistent landmarks at the birth-scar-distal and -proximal poles that involve the proteins Bud8p and Bud9p, respectively. Rax1p and Rax2p also appear to function specifically in bipolar budding, and we report here a further characterization of these proteins and of their interactions with Bud8p and Bud9p. Rax1p and Rax2p both appear to be integral membrane proteins. Although commonly used programs predict different topologies for Rax2p, glycosylation studies indicate that it has a type I orientation, with its long N-terminal domain in the extracytoplasmic space. Analysis of rax1 and rax2 mutant budding patterns indicates that both proteins are involved in selecting bud sites at both the distal and proximal poles of daughter cells as well as near previously used division sites on mother cells. Consistent with this, GFP-tagged Rax1p and Rax2p were both observed at the distal pole as well as at the division site on both mother and daughter cells; localization to the division sites was persistent through multiple cell cycles. Localization of Rax1p and Rax2p was interdependent, and biochemical studies showed that these proteins could be copurified from yeast. Bud8p and Bud9p could also be copurified with Rax1p, and localization studies provided further evidence of interactions. Localization of Rax1p and Rax2p to the bud tip and distal pole depended on Bud8p, and normal localization of Bud8p was partially dependent on Rax1p and Rax2p. Although localization of Rax1p and Rax2p to the division site did not appear to depend on Bud9p, normal localization of Bud9p appeared largely or entirely dependent on Rax1p and Rax2p. Taken together, the results indicate that Rax1p and Rax2p interact closely with each other and with Bud8p and Bud9p in the establishment and/or maintenance of the cortical landmarks for bipolar budding.

The role of Cdc42p GTPase-activating proteins in assembly of the septin ring in yeastMOLECULAR BIOLOGY OF THE CELLCaviston, J. P., Longtine, M., Pringle, J. R., Bi, E.2003; 14 (10): 4051-4066

Abstract

The septins are a conserved family of GTP-binding, filament-forming proteins. In the yeast Saccharomyces cerevisiae, the septins form a ring at the mother-bud neck that appears to function primarily by serving as a scaffold for the recruitment of other proteins to the neck, where they participate in cytokinesis and a variety of other processes. Formation of the septin ring depends on the Rho-type GTPase Cdc42p but appears to be independent of the actin cytoskeleton. In this study, we investigated further the mechanisms of septin-ring formation. Fluorescence-recovery-after-photobleaching (FRAP) experiments indicated that the initial septin structure at the presumptive bud site is labile (exchanges subunits freely) but that it is converted into a stable ring as the bud emerges. Mutants carrying the cdc42V36G allele or lacking two or all three of the known Cdc42p GTPase-activating proteins (GAPs: Bem3p, Rga1p, and Rga2p) could recruit the septins to the cell cortex but were blocked or delayed in forming a normal septin ring and had accompanying morphogenetic defects. These phenotypes were dramatically enhanced in mutants that were also defective in Cla4p or Gin4p, two protein kinases previously shown to be important for normal septin-ring formation. The Cdc42p GAPs colocalized with the septins both early and late in the cell cycle, and overexpression of the GAPs could suppress the septin-organization and morphogenetic defects of temperature-sensitive septin mutants. Taken together, the data suggest that formation of the mature septin ring is a process that consists of at least two distinguishable steps, recruitment of the septin proteins to the presumptive bud site and their assembly into the stable septin ring. Both steps appear to depend on Cdc42p, whereas the Cdc42p GAPs and the other proteins known to promote normal septin-ring formation appear to function in a partially redundant manner in the assembly step. In addition, because the eventual formation of a normal septin ring in a cdc42V36G or GAP mutant was invariably accompanied by a switch from an abnormally elongated to a more normal bud morphology distal to the ring, it appears that the septin ring plays a direct role in determining the pattern of bud growth.

Abstract

There are 10 known mammalian septin genes, some of which produce multiple splice variants. The current nomenclature for the genes and gene products is very confusing, with several different names having been given to the same gene product and distinct names given to splice variants of the same gene. Moreover, some names are based on those of yeast or Drosophila septins that are not the closest homologues. Therefore, we suggest that the mammalian septin field adopt a common nomenclature system, based on that adopted by the Mouse Genomic Nomenclature Committee and accepted by the Human Genome Organization Gene Nomenclature Committee. The human and mouse septin genes will be named SEPT1-SEPT10 and Sept1-Sept10, respectively. Splice variants will be designated by an underscore followed by a lowercase "v" and a number, e.g., SEPT4_v1.

Abstract

In the budding yeast Saccharomyces cerevisiae, the Cdc3p, Cdc10p, Cdc11p, Cdc12p, and Sep7p/Shs1p septins assemble early in the cell cycle in a ring that marks the future cytokinetic site. The septins appear to be major structural components of a set of filaments at the mother-bud neck and function as a scaffold for recruiting proteins involved in cytokinesis and other processes. We isolated a novel gene, BNI5, as a dosage suppressor of the cdc12-6 growth defect. Overexpression of BNI5 also suppressed the growth defects of cdc10-1, cdc11-6, and sep7Delta strains. Loss of BNI5 resulted in a cytokinesis defect, as evidenced by the formation of connected cells with shared cytoplasms, and deletion of BNI5 in a cdc3-6, cdc10-1, cdc11-6, cdc12-6, or sep7Delta mutant strain resulted in enhanced defects in septin localization and cytokinesis. Bni5p localizes to the mother-bud neck in a septin-dependent manner shortly after bud emergence and disappears from the neck approximately 2 to 3 min before spindle disassembly. Two-hybrid, in vitro binding, and protein-localization studies suggest that Bni5p interacts with the N-terminal domain of Cdc11p, which also appears to be sufficient for the localization of Cdc11p, its interaction with other septins, and other critical aspects of its function. Our data suggest that the Bni5p-septin interaction is important for septin ring stability and function, which is in turn critical for normal cytokinesis.

Abstract

The septins are a family of proteins involved in cytokinesis and other aspects of cell-cortex organization. In a two-hybrid screen designed to identify septin-interacting proteins in Drosophila, we isolated several genes, including homologues (Dmuba2 and Dmubc9) of yeast UBA2 and UBC9. Yeast Uba2p and Ubc9p are involved in the activation and conjugation, respectively, of the ubiquitin-like protein Smt3p/SUMO, which becomes conjugated to a variety of proteins through this pathway. Uba2p functions together with a second protein, Aos1p. We also cloned and characterized the Drosophila homologues of AOS1 (Dmaos1) and SMT3 (Dmsmt3). Our biochemical data suggest that DmUba2/DmAos1 and DmUbc9 indeed act as activating and conjugating enzymes for DmSmt3, implying that this protein-conjugation pathway is well conserved in Drosophila. Immunofluorescence studies showed that DmUba2 shuttles between the embryonic cortex and nuclei during the syncytial blastoderm stage. In older embryos, DmUba2 and DmSmt3 are both concentrated in the nuclei during interphase but dispersed throughout the cells during mitosis, with DmSmt3 also enriched on the chromosomes during mitosis. These data suggest that DmSmt3 could modify target proteins both inside and outside the nuclei. We did not observe any concentration of DmUba2 at sites where the septins are concentrated, and we could not detect DmSmt3 modification of the three Drosophila septins tested. However, we did observe DmSmt3 localization to the midbody during cytokinesis both in tissue-culture cells and in embryonic mitotic domains, suggesting that DmSmt3 modification of septins and/or other midzone proteins occurs during cytokinesis in Drosophila.

Abstract

In Saccharomyces cerevisiae, Bud8p and Bud9p are homologous plasma membrane glycoproteins that appear to mark the distal and proximal cell poles, respectively, as potential sites for budding in the bipolar pattern. Here we provide evidence that Bud8p is delivered to the presumptive bud site (and thence to the distal pole of the bud) just before bud emergence, and that Bud9p is delivered to the bud side of the mother-bud neck (and thence to the proximal pole of the daughter cell) after activation of the mitotic exit network, just before cytokinesis. Like the delivery of Bud8p, that of Bud9p is actin dependent; unlike the delivery of Bud8p, that of Bud9p is also septin dependent. Interestingly, although the transcription of BUD8 and BUD9 appears to be cell cycle regulated, the abundance of BUD8 mRNA peaks in G2/M and that of BUD9 mRNA peaks in late G1, suggesting that the translation and/or delivery to the cell surface of each protein is delayed and presumably also cell cycle regulated. The importance of time of transcription in localization is supported by promoter-swap experiments: expression of Bud8p from the BUD9 promoter leads to its localization predominantly to the sites typical for Bud9p, and vice versa. Moreover, expression of Bud8p from the BUD9 promoter fails to rescue the budding-pattern defect of a bud8 mutant but fully rescues that of a bud9 mutant. However, although expression of Bud9p from the BUD8 promoter fails to rescue a bud9 mutant, it also rescues only partially the budding-pattern defect of a bud8 mutant, suggesting that some feature(s) of the Bud8p protein is also important for Bud8p function. Experiments with chimeric proteins suggest that the critical element(s) is somewhere in the extracytoplasmic domain of Bud8p.

Abstract

A specialized cortical domain is organized by the septins at the necks of budding yeast cells. Recent findings suggest that this domain serves as a diffusion barrier and also as a local cell-shape sensor. We review these findings along with what is known about the organization of the septin cortex and its regulation during the cell cycle.

Abstract

Many genes required for cell polarity development in budding yeast have been identified and arranged into a functional hierarchy. Core elements of the hierarchy are widely conserved, underlying cell polarity development in diverse eukaryotes. To enumerate more fully the protein-protein interactions that mediate cell polarity development, and to uncover novel mechanisms that coordinate the numerous events involved, we carried out a large-scale two-hybrid experiment. 68 Gal4 DNA binding domain fusions of yeast proteins associated with the actin cytoskeleton, septins, the secretory apparatus, and Rho-type GTPases were used to screen an array of yeast transformants that express approximately 90% of the predicted Saccharomyces cerevisiae open reading frames as Gal4 activation domain fusions. 191 protein-protein interactions were detected, of which 128 had not been described previously. 44 interactions implicated 20 previously uncharacterized proteins in cell polarity development. Further insights into possible roles of 13 of these proteins were revealed by their multiple two-hybrid interactions and by subcellular localization. Included in the interaction network were associations of Cdc42 and Rho1 pathways with proteins involved in exocytosis, septin organization, actin assembly, microtubule organization, autophagy, cytokinesis, and cell wall synthesis. Other interactions suggested direct connections between Rho1- and Cdc42-regulated pathways; the secretory apparatus and regulators of polarity establishment; actin assembly and the morphogenesis checkpoint; and the exocytic and endocytic machinery. In total, a network of interactions that provide an integrated response of signaling proteins, the cytoskeleton, and organelles to the spatial cues that direct polarity development was revealed.

Abstract

The bipolar budding pattern of a/alpha Saccharomyces cerevisiae cells appears to depend on persistent spatial markers in the cell cortex at the two poles of the cell. Previous analysis of mutants with specific defects in bipolar budding identified BUD8 and BUD9 as potentially encoding components of the markers at the poles distal and proximal to the birth scar, respectively. Further genetic analysis reported here supports this hypothesis. Mutants deleted for BUD8 or BUD9 grow normally but bud exclusively from the proximal and distal poles, respectively, and the double-mutant phenotype suggests that the bipolar budding pathway has been totally disabled. Moreover, overexpression of these genes can cause either an increased bias for budding at the distal (BUD8) or proximal (BUD9) pole or a randomization of bud position, depending on the level of expression. The structures and localizations of Bud8p and Bud9p are also consistent with their postulated roles as cortical markers. Both proteins appear to be integral membrane proteins of the plasma membrane, and they have very similar overall structures, with long N-terminal domains that are both N- and O-glycosylated followed by a pair of putative transmembrane domains surrounding a short hydrophilic domain that is presumably cytoplasmic. The putative transmembrane and cytoplasmic domains of the two proteins are very similar in sequence. When Bud8p and Bud9p were localized by immunofluorescence and tagging with GFP, each protein was found predominantly in the expected location, with Bud8p at presumptive bud sites, bud tips, and the distal poles of daughter cells and Bud9p at the necks of large-budded cells and the proximal poles of daughter cells. Bud8p localized approximately normally in several mutants in which daughter cells are competent to form their first buds at the distal pole, but it was not detected in a bni1 mutant, in which such distal-pole budding is lost. Surprisingly, Bud8p localization to the presumptive bud site and bud tip also depends on actin but is independent of the septins.

Abstract

Eukaryotic cells contain many actin-interacting proteins, including the alpha-actinins and the fimbrins, both of which have actin cross-linking activity in vitro. We report here the identification and characterization of both an alpha-actinin-like protein (Ain1p) and a fimbrin (Fim1p) in the fission yeast Schizosaccharomyces pombe. Ain1p localizes to the actomyosin-containing medial ring in an F-actin-dependent manner, and the Ain1p ring contracts during cytokinesis. ain1 deletion cells have no obvious defects under normal growth conditions but display severe cytokinesis defects, associated with defects in medial-ring and septum formation, under certain stress conditions. Overexpression of Ain1p also causes cytokinesis defects, and the ain1 deletion shows synthetic effects with other mutations known to affect medial-ring positioning and/or organization. Fim1p localizes both to the cortical actin patches and to the medial ring in an F-actin-dependent manner, and several lines of evidence suggest that Fim1p is involved in polarization of the actin cytoskeleton. Although a fim1 deletion strain has no detectable defect in cytokinesis, overexpression of Fim1p causes a lethal cytokinesis defect associated with a failure to form the medial ring and concentrate actin patches at the cell middle. Moreover, an ain1 fim1 double mutant has a synthetical-lethal defect in medial-ring assembly and cell division. Thus, Ain1p and Fim1p appear to have an overlapping and essential function in fission yeast cytokinesis. In addition, protein-localization and mutant-phenotype data suggest that Fim1p, but not Ain1p, plays important roles in mating and in spore formation.

Abstract

The septins are a conserved family of proteins that are involved in cytokinesis and other aspects of cell-surface organization. In Drosophila melanogaster, null mutations in the pnut septin gene are recessive lethal, but homozygous pnut mutants complete embryogenesis and survive until the pupal stage. Because the completion of cellularization and other aspects of early development seemed likely to be due to maternally contributed Pnut product, we attempted to generate embryos lacking the maternal contribution in order to explore the roles of Pnut in these processes. We used two methods, the production of germline clones homozygous for a pnut mutation and the rescue of pnut homozygous mutant flies by a pnut(+) transgene under control of the hsp70 promoter. Remarkably, the pnut germline-clone females produced eggs, indicating that stem-cell and cystoblast divisions in the female germline do not require Pnut. Moreover, the Pnut-deficient embryos obtained by either method completed early syncytial development and began cellularization of the embryo normally. However, during the later stages of cellularization, the organization of the actin cytoskeleton at the leading edge of the invaginating furrows became progressively more abnormal, and the embryos displayed widespread defects in cell and embryo morphology beginning at gastrulation. Examination of two other septins showed that Sep1 was not detectable at the cellularization front in the Pnut-deficient embryos, whereas Sep2 was still present in normal levels. Thus, it is possible that Sep2 (perhaps in conjunction with other septins such as Sep4 and Sep5) fulfills an essential septin role during the organization and initial ingression of the cellularization furrow even in the absence of Pnut and Sep1. Together, the results suggest that some cell-division events in Drosophila do not require septin function, that there is functional differentiation among the Drosophila septins, or both.

Abstract

Saccharomyces cerevisiae septin mutants have pleiotropic defects, which include the formation of abnormally elongated buds. This bud morphology results at least in part from a cell cycle delay imposed by the Cdc28p-inhibitory kinase Swe1p. Mutations in three other genes (GIN4, encoding a kinase related to the Schizosaccharomyces pombe mitotic inducer Nim1p; CLA4, encoding a p21-activated kinase; and NAP1, encoding a Clb2p-interacting protein) also produce perturbations of septin organization associated with an Swe1p-dependent cell cycle delay. The effects of gin4, cla4, and nap1 mutations are additive, indicating that these proteins promote normal septin organization through pathways that are at least partially independent. In contrast, mutations affecting the other two Nim1p-related kinases in S. cerevisiae, Hsl1p and Kcc4p, produce no detectable effect on septin organization. However, deletion of HSL1, but not of KCC4, did produce a cell cycle delay under some conditions; this delay appears to reflect a direct role of Hsl1p in the regulation of Swe1p. As shown previously, Swe1p plays a central role in the morphogenesis checkpoint that delays the cell cycle in response to defects in bud formation. Swe1p is localized to the nucleus and to the daughter side of the mother bud neck prior to its degradation in G(2)/M phase. Both the neck localization of Swe1p and its degradation require Hsl1p and its binding partner Hsl7p, both of which colocalize with Swe1p at the daughter side of the neck. This localization is lost in mutants with perturbed septin organization, suggesting that the release of Hsl1p and Hsl7p from the neck may reduce their ability to inactivate Swe1p and thus contribute to the G(2) delay observed in such mutants. In contrast, treatments that perturb actin organization have little effect on Hsl1p and Hsl7p localization, suggesting that such treatments must stabilize Swe1p by another mechanism. The apparent dependence of Swe1p degradation on localization of the Hsl1p-Hsl7p-Swe1p module to a site that exists only in budded cells may constitute a mechanism for deactivating the morphogenesis checkpoint when it is no longer needed (i.e., after a bud has formed).

Abstract

In the yeast Saccharomyces cerevisiae, Cdc24p functions at least in part as a guanine-nucleotide-exchange factor for the Rho-family GTPase Cdc42p. A genetic screen designed to identify possible additional targets of Cdc24p instead identified two previously known genes, MSB1 and CLA4, and one novel gene, designated MSB3, all of which appear to function in the Cdc24p-Cdc42p pathway. Nonetheless, genetic evidence suggests that Cdc24p may have a function that is distinct from its Cdc42p guanine-nucleotide-exchange factor activity; in particular, overexpression of CDC42 in combination with MSB1 or a truncated CLA4 in cells depleted for Cdc24p allowed polarization of the actin cytoskeleton and polarized cell growth, but not successful cell proliferation. MSB3 has a close homologue (designated MSB4) and two more distant homologues (MDR1 and YPL249C) in S. cerevisiae and also has homologues in Schizosaccharomyces pombe, Drosophila (pollux), and humans (the oncogene tre17). Deletion of either MSB3 or MSB4 alone did not produce any obvious phenotype, and the msb3 msb4 double mutant was viable. However, the double mutant grew slowly and had a partial disorganization of the actin cytoskeleton, but not of the septins, in a fraction of cells that were larger and rounder than normal. Like Cdc42p, both Msb3p and Msb4p localized to the presumptive bud site, the bud tip, and the mother-bud neck, and this localization was Cdc42p dependent. Taken together, the data suggest that Msb3p and Msb4p may function redundantly downstream of Cdc42p, specifically in a pathway leading to actin organization. From previous work, the BNI1, GIC1, and GIC2 gene products also appear to be involved in linking Cdc42p to the actin cytoskeleton. Synthetic lethality and multicopy suppression analyses among these genes, MSB, and MSB4, suggest that the linkage is accomplished by two parallel pathways, one involving Msb3p, Msb4p, and Bni1p, and the other involving Gic1p and Gic2p. The former pathway appears to be more important in diploids and at low temperatures, whereas the latter pathway appears to be more important in haploids and at high temperatures.

Abstract

In animal and fungal cells, cytokinesis involves an actomyosin ring that forms and contracts at the division plane. Important new details have emerged concerning the composition, assembly, and dynamics of these contractile rings. In addition, recent advances suggest that targeted membrane addition is a central feature of cytokinesis in animal cells - as it is in fungi and plants - and the coordination of actomyosin ring function with targeted exocytosis at the cleavage plane is being explored. Important new information has also emerged about the spatial and temporal regulation of cytokinesis, especially in relation to the function of the spindle midzone in animal cells and the control of cytokinesis by GTPase systems.

Abstract

In Saccharomyces cerevisiae, the Wee1 family kinase Swe1p is normally stable during G(1) and S phases but is unstable during G(2) and M phases due to ubiquitination and subsequent degradation. However, perturbations of the actin cytoskeleton lead to a stabilization and accumulation of Swe1p. This response constitutes part of a morphogenesis checkpoint that couples cell cycle progression to proper bud formation, but the basis for the regulation of Swe1p degradation by the morphogenesis checkpoint remains unknown. Previous studies have identified a protein kinase, Hsl1p, and a phylogenetically conserved protein of unknown function, Hsl7p, as putative negative regulators of Swe1p. We report here that Hsl1p and Hsl7p act in concert to target Swe1p for degradation. Both proteins are required for Swe1p degradation during the unperturbed cell cycle, and excess Hsl1p accelerates Swe1p degradation in the G(2)-M phase. Hsl1p accumulates periodically during the cell cycle and promotes the periodic phosphorylation of Hsl7p. Hsl7p can be detected in a complex with Swe1p in cell lysates, and the overexpression of Hsl7p or Hsl1p produces an effective override of the G(2) arrest imposed by the morphogenesis checkpoint. These findings suggest that Hsl1p and Hsl7p interact directly with Swe1p to promote its recognition by the ubiquitination complex, leading ultimately to its destruction.

Abstract

The fission yeast Schizosaccharomyces pombe divides symmetrically using a medial F-actin- based contractile ring to produce equal-sized daughter cells. Mutants defective in two previously described genes, mid1 and pom1, frequently divide asymmetrically. Here we present the identification of three new temperature-sensitive mutants defective in localization of the division plane. All three mutants have mutations in the polo kinase gene, plo1, and show defects very similar to those of mid1 mutants in both the placement and organization of the medial ring. In both cases, ring formation is frequently initiated near the cell poles, indicating that Mid1p and Plo1p function in recruiting medial ring components to the cell center. It has been reported previously that during mitosis Mid1p becomes hyperphosphorylated and relocates from the nucleus to a medial ring. Here we show that Mid1p first forms a diffuse cortical band during spindle formation and then coalesces into a ring before anaphase. Plo1p is required for Mid1p to exit the nucleus and form a ring, and Pom1p is required for proper placement of the Mid1p ring. Upon overexpression of Plo1p, Mid1p exits the nucleus prematurely and displays a reduced mobility on gels similar to that of the hyperphosphorylated form observed previously in mitotic cells. Genetic and two-hybrid analyses suggest that Plo1p and Mid1p act in a common pathway distinct from that involving Pom1p. Plo1p localizes to the spindle pole bodies and spindles of mitotic cells and also to the medial ring at the time of its formation. Taken together, the data indicate that Plo1p plays a role in the positioning of division sites by regulating Mid1p. Given its previously known functions in mitosis and the timing of cytokinesis, Plo1p is thus implicated as a key molecule in the spatial and temporal coordination of cytokinesis with mitosis.

Abstract

To identify septin-interacting proteins in Saccharomyces cerevisiae, we screened for mutations that are synthetically lethal with a cdc12 septin mutation. One of the genes identified was GIN4, which encodes a protein kinase related to Hsl1p/Nik1p and Ycl024Wp in S. cerevisiae and to Nim1p/Cdr1p and Cdr2p in Schizosaccharomyces pombe. The Gin4p kinase domain displayed a two-hybrid interaction with the COOH-terminal portion of the Cdc3p septin, and Gin4p colocalized with the septins at the mother-bud neck. This localization depended on the septins and on the COOH-terminal (nonkinase) region of Gin4p, and overproduction of this COOH-terminal region led to a loss of septin organization and associated morphogenetic defects. We detected no effect of deleting YCL024W, either alone or in combination with deletion of GIN4. Deletion of GIN4 was not lethal but led to a striking reorganization of the septins accompanied by morphogenetic abnormalities and a defect in cell separation; however, remarkably, cytokinesis appeared to occur efficiently. Two other proteins that localize to the neck in a septin-dependent manner showed similar reorganizations and also appeared to remain largely functional. The septin organization observed in gin4Delta vegetative cells resembles that seen normally in cells responding to mating pheromone, and no Gin4p was detected in association with the septins in such cells. The organization of the septins observed in gin4Delta cells and in cells responding to pheromone appears to support some aspects of the model for septin organization suggested previously by Field et al. (Field, C.M., O. Al-Awar, J. Rosenblatt, M.L. Wong, B. Alberts, and T.J. Mitchison. 1996. J. Cell Biol. 133:605-616).

Abstract

The septins are a family of proteins required for cytokinesis in a number of eukaryotic cell types. In budding yeast, these proteins are thought to be the structural components of a filament system present at the mother-bud neck, called the neck filaments. In this study, we report the isolation of a protein complex containing the yeast septins Cdc3p, Cdc10p, Cdc11p, and Cdc12p that is capable of forming long filaments in vitro. To investigate the relationship between these filaments and the neck filaments, we purified septin complexes from cells deleted for CDC10 or CDC11. These complexes were not capable of the polymerization exhibited by wild-type preparations, and analysis of the neck region by electron microscopy revealed that the cdc10Delta and cdc11Delta cells did not contain detectable neck filaments. These results strengthen the hypothesis that the septins are the major structural components of the neck filaments. Surprisingly, we found that septin dependent processes like cytokinesis and the localization of Bud4p to the neck still occurred in cdc10Delta cells. This suggests that the septins may be able to function in the absence of normal polymerization and the formation of a higher order filament structure.

Abstract

In Saccharomyces cerevisiae, the mother cell and bud are connected by a narrow neck. The mechanism by which this neck is closed during cytokinesis has been unclear. Here we report on the role of a contractile actomyosin ring in this process. Myo1p (the only type II myosin in S. cerevisiae) forms a ring at the presumptive bud site shortly before bud emergence. Myo1p ring formation depends on the septins but not on F-actin, and preexisting Myo1p rings are stable when F-actin is depolymerized. The Myo1p ring remains in the mother-bud neck until the end of anaphase, when a ring of F-actin forms in association with it. The actomyosin ring then contracts to a point and disappears. In the absence of F-actin, the Myo1p ring does not contract. After ring contraction, cortical actin patches congregate at the mother-bud neck, and septum formation and cell separation rapidly ensue. Strains deleted for MYO1 are viable; they fail to form the actin ring but show apparently normal congregation of actin patches at the neck. Some myo1Delta strains divide nearly as efficiently as wild type; other myo1Delta strains divide less efficiently, but it is unclear whether the primary defect is in cytokinesis, septum formation, or cell separation. Even cells lacking F-actin can divide, although in this case division is considerably delayed. Thus, the contractile actomyosin ring is not essential for cytokinesis in S. cerevisiae. In its absence, cytokinesis can still be completed by a process (possibly localized cell-wall synthesis leading to septum formation) that appears to require septin function and to be facilitated by F-actin.

Abstract

To determine if the attached cells formed in Myosin II-deficient Saccharomyces cerevisiae result from deficient chitinase 1 (CTS1) expression, the activity of chitinase 1 was assayed. Secretion of this enzyme was not prevented by a MYO1 gene deficiency, and soluble and cell wall-associated Cts1p activity were increased approximately 5-fold and 20-fold, respectively, in these cells. The increase in soluble activity was correlated with an increase in enzyme levels. Likewise, intracellular chitinase activity was increased approximately 22-fold, and the chitin content of cell walls was elevated 2-fold. These data suggest that the origin of myo1-associated phenotypes is not due to deficient chitinase expression and may instead be due to a deregulation of cell wall metabolism in these cells.

Abstract

We describe a straightforward PCR-based approach to the deletion, tagging, and overexpression of genes in their normal chromosomal locations in the fission yeast Schizosaccharomyces pombe. Using this approach and the S. pombe ura4+ gene as a marker, nine genes were deleted with efficiencies of homologous integration ranging from 6 to 63%. We also constructed a series of plasmids containing the kanMX6 module, which allows selection of G418-resistant cells and thus provides a new heterologous marker for use in S. pombe. The modular nature of these constructs allows a small number of PCR primers to be used for a wide variety of gene manipulations, including deletion, overexpression (using the regulatable nmt1 promoter), C- or N-terminal protein tagging (with HA, Myc, GST, or GFP), and partial C- or N-terminal deletions with or without tagging. Nine genes were manipulated using these kanMX6 constructs as templates for PCR. The PCR primers included 60 to 80 bp of flanking sequences homologous to target sequences in the genome. Transformants were screened for homologous integration by PCR. In most cases, the efficiency of homologous integration was > or = 50%, and the lowest efficiency encountered was 17%. The methodology and constructs described here should greatly facilitate analysis of gene function in S. pombe.

Abstract

An important recent advance in the functional analysis of Saccharomyces cerevisiae genes is the development of the one-step PCR-mediated technique for deletion and modification of chromosomal genes. This method allows very rapid gene manipulations without requiring plasmid clones of the gene of interest. We describe here a new set of plasmids that serve as templates for the PCR synthesis of fragments that allow a variety of gene modifications. Using as selectable marker the S. cerevisiae TRP1 gene or modules containing the heterologous Schizosaccharomyces pombe his5+ or Escherichia coli kan(r) gene, these plasmids allow gene deletion, gene overexpression (using the regulatable GAL1 promoter), C- or N-terminal protein tagging [with GFP(S65T), GST, or the 3HA or 13Myc epitope], and partial N- or C-terminal deletions (with or without concomitant protein tagging). Because of the modular nature of the plasmids, they allow efficient and economical use of a small number of PCR primers for a wide variety of gene manipulations. Thus, these plasmids should further facilitate the rapid analysis of gene function in S. cerevisiae.

Abstract

Schizosaccharomyces pombe cells have a well-defined pattern of polarized growth at the cell ends during interphase and divide symmetrically into two equal-sized daughter cells. We identified a gene, pom1, that provides positional information for both growth and division in S. pombe. pom1 mutants form functioning growth zones and division septa but show several abnormalities: (1) After division, cells initiate growth with equal frequencies from either the old or the new end; (2) most cells never switch to bipolar growth but instead grow exclusively at the randomly chosen end; (3) some cells mislocalize their growth axis altogether, leading to the formation of angled and branched cells; and (4) many cells misplace and/or misorient their septa, leading to asymmetric cell division. pom1 encodes a putative protein kinase that is concentrated at the new cell end during interphase, at both cell ends during mitosis, and at the septation site after mitosis. Small amounts of Pom1p are also found at the old cell end during interphase and associated with the actin ring during mitosis. Pom1p localization to the cell ends is independent of actin but requires microtubules and Tea1p. pom1 mutations are synthetically lethal with several other mutations that affect cytokinesis and/or the actin or microtubule cytoskeleton. Thus, Pom1p may position the growth and cytokinesis machineries by interaction with both the actin and microtubule cytoskeletons.

Abstract

Just before bud emergence, a Saccharomyces cerevisiae cell forms a ring of chitin in its cell wall; this ring remains at the base of the bud as the bud grows and ultimately forms part of the bud scar marking the division site on the mother cell. The chitin ring seems to be formed largely or entirely by chitin synthase III, one of the three known chitin synthases in S. cerevisiae. The chitin ring does not form normally in temperature-sensitive mutants defective in any of four septins, a family of proteins that are constituents of the "neck filaments" that lie immediately subjacent to the plasma membrane in the mother-bud neck. In addition, a synthetic-lethal interaction was found between cdc12-5, a temperature-sensitive septin mutation, and a mutant allele of CHS4, which encodes an activator of chitin synthase III. Two-hybrid analysis revealed no direct interaction between the septins and Chs4p but identified a novel gene, BNI4, whose product interacts both with Chs4p and Cdc10p and with one of the septins, Cdc10p; this analysis also revealed an interaction between Chs4p and Chs3p, the catalytic subunit of chitin synthase III. Bni4p has no known homologues; it contains a predicted coiled-coil domain, but no other recognizable motifs. Deletion of BNI4 is not lethal, but causes delocalization of chitin deposition and aberrant cellular morphology. Overexpression of Bni4p also causes delocalization of chitin deposition and produces a cellular morphology similar to that of septin mutants. Immunolocalization experiments show that Bni4p localizes to a ring at the mother-bud neck that lies predominantly on the mother-cell side (corresponding to the predominant site of chitin deposition). This localization depends on the septins but not on Chs4p or Chs3p. A GFP-Chs4p fusion protein also localizes to a ring at the mother-bud neck on the mother-cell side. This localization is dependent on the septins, Bni4p, and Chs3p. Chs3p, whose normal localization is similar to that of Chs4p, does not localize properly in bni4, chs4, or septin mutant strains or in strains that accumulate excess Bni4p. In contrast, localization of the septins is essentially normal in bni4, chs4, and chs3 mutant strains and in strains that accumulate excess Bni4p. Taken together, these results suggest that the normal localization of chitin synthase III activity is achieved by assembly of a complex in which Chs3p is linked to the septins via Chs4p and Bni4p.

Two active states of the Ras-related Bud1/Rsr1 protein bind to different effectors to determine yeast cell polarityPROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICAPark, H. O., Bi, E. F., Pringle, J. R., Herskowitz, I.1997; 94 (9): 4463-4468

Abstract

Cells of budding yeast organize their cytoskeleton in a highly polarized manner during vegetative growth. Selection of a site for polarization requires a group of proteins including a Ras-like GTPase, Bud1, and its regulators. Another group of proteins, which includes a Rho-like GTPase (Cdc42), its guanine nucleotide exchange factor (Cdc24), and Bem1, is necessary for organization of the actin cytoskeleton and for cell polarization. We have proposed previously that the Bud1 protein, through its GTPase cycle, determines the localization of one or more of the cell polarity proteins to the bud site. Herein we demonstrate that Bud1 directly interacts with Cdc24 and Bem1: Bud1 in its GTP-bound form associates preferentially with Cdc24, whereas the GDP-bound form of Bud1 associates with Bem1. We also present subcellular fractionation data for Bud1 that is consistent with the idea that Bud1 can travel between the site for budding on the plasma membrane and the cytosol. We propose that Bud1 can exist in two active states for association with different partners and that the switch from Bud1-GTP to Bud1-GDP provides a regulatory device for ordered assembly of a macromolecular complex at the bud site.

Abstract

The Saccharomyces cerevisiae BNI1 gene product (Bni1p) is a member of the formin family of proteins, which participate in cell polarization, cytokinesis, and vertebrate limb formation. During mating pheromone response, bni1 mutants showed defects both in polarized morphogenesis and in reorganization of the underlying actin cytoskeleton. In two-hybrid experiments, Bni1p formed complexes with the activated form of the Rho-related guanosine triphosphatase Cdc42p, with actin, and with two actin-associated proteins, profilin and Bud6p (Aip3p). Both Bni1p and Bud6p (like Cdc42p and actin) localized to the tips of mating projections. Bni1p may function as a Cdc42p target that links the pheromone response pathway to the actin cytoskeleton.

Abstract

A search for Saccharomyces cerevisiae proteins that interact with actin in the two-hybrid system and a screen for mutants that affect the bipolar budding pattern identified the same gene, AIP3/BUD6. This gene is not essential for mitotic growth but is necessary for normal morphogenesis. MATa/alpha daughter cells lacking Aip3p place their first buds normally at their distal poles but choose random sites for budding in subsequent cell cycles. This suggests that actin and associated proteins are involved in placing the bipolar positional marker at the division site but not at the distal tip of the daughter cell. In addition, although aip3 mutant cells are not obviously defective in the initial polarization of the cytoskeleton at the time of bud emergence, they appear to lose cytoskeletal polarity as the bud enlarges, resulting in the formation of cells that are larger and rounder than normal. aip3 mutant cells also show inefficient nuclear migration and nuclear division, defects in the organization of the secretory system, and abnormal septation, all defects that presumably reflect the involvement of Aip3p in the organization and/or function of the actin cytoskeleton. The sequence of Aip3p is novel but contains a predicted coiled-coil domain near its C terminus that may mediate the observed homo-oligomerization of the protein. Aip3p shows a distinctive localization pattern that correlates well with its likely sites of action: it appears at the presumptive bud site prior to bud emergence, remains near the tips of small bund, and forms a ring (or pair of rings) in the mother-bud neck that is detectable early in the cell cycle but becomes more prominent prior to cytokinesis. Surprisingly, the localization of Aip3p does not appear to require either polarized actin or the septin proteins of the neck filaments.

Abstract

Regulation of cell cycle progression occurs in part through the targeted degradation of both activating and inhibitory subunits of the cyclin-dependent kinases. During G1, CDC4, encoding a WD-40 repeat protein, and CDC34, encoding a ubiquitin-conjugating enzyme, are involved in the destruction of these regulators. Here we describe evidence indicating that CDC53 also is involved in this process. Mutations in CDC53 cause a phenotype indistinguishable from those of cdc4 and cdc34 mutations, numerous genetic interactions are seen between these genes, and the encoded proteins are found physically associated in vivo. Cdc53p defines a large family of proteins found in yeasts, nematodes, and humans whose molecular functions are uncharacterized. These results suggest a role for this family of proteins in regulating cell cycle proliferation through protein degradation.

SPR28, a sixth member of the septin gene family in Saccharomyces cerevisiae that is expressed specifically in sporulating cellsMICROBIOLOGY-SGMDEVIRGILIO, C., DeMarini, D. J., Pringle, J. R.1996; 142: 2897-2905

Abstract

The septins are a recently recognized family of proteins that are present in a wide variety of fungal and animal cells, where they are involved in cytokinesis and apparently in other processes involving the organization of the cell surface. Five previously described Saccharomyces cerevisiae septins are associated with the neck filaments of vegetative cells and/or with the developing prospore wall of sporulating cells. We report here the characterization of SPR28, a sixth member of the S. cerevisiae septin gene family whose existence was revealed by the yeast genome project. Analysis of mRNA levels showed that SPR28 is a new member of the group of "late genes' that are expressed at high levels during the meiotic divisions and ascospore formation. The septin it encodes, Spr28p, exhibited specific two-hybrid interactions with itself and with three other septins that are expressed in sporulating cells. Consistent with these results, an Spr28p-green fluorescent protein fusion was induced during meiosis I and appeared to be associated with the developing prospore walls. Deletion of SPR28 in either a wild-type or an spr3 delta background produced no obvious abnormalities in vegetative cells and had little or no effect on sporulation, suggesting that the septins have redundant roles during spore formation.

Abstract

A genetic screen for GTPase-activating proteins (GAPs) or other negative regulators of the Rac/Rho family GTPase Cdc42p in Saccharomyces cerevisiae identified ZDS1, a gene encoding a protein of 915 amino acids. Sequence from the yeast genome project identified a homolog, ZDS2, whose predicted product of 942 amino acids is 38% identical in sequence to Zds1p. Zds1p and Zds2p have no detectable homology to known Rho-GAPs or to other known proteins. However, by several assays, it appears that overexpression of either Zds1p or Zds2p decreases the level of Cdc42p activity. Deletion analysis also suggests that Zds1p and Zds2p are at least partially overlapping in function. Deletion of ZDS2 produced no obvious phenotype, and deletion of ZDS1 produced no obvious phenotype other than a mild effect on cell shape. However, the zds1 zds2 double mutant grew slowly with an apparent mitotic delay and produced elongated cells and buds with other evidence of abnormal morphogenesis. A glutathione S-transferase-Zds1p fusion protein that fully complemented the double mutant localized to presumptive bud sites and the tips of small buds. The similarity of this localization to that of Cdc42p suggests that Zds1p may interact directly with Cdc42p. As ZDS1 and ZDS2 have recently been identified also by numerous other groups studying a wide range of biological phenomena, the roles of Cdc42p in intracellular signaling may be more diverse than has previously been appreciated.

Abstract

The gene CAL1 (also known as CDC43) of Saccharomyces cerevisiae encodes the beta subunit of geranylgeranyl transferase I (GGTase I), which modifies several small GTPases. Biochemical analyses of the mutant enzymes encoded by cal1-1, and cdc43-2 to cdc43-7, expressed in bacteria, have shown that all of the mutant enzymes possess reduced activity, and that none shows temperature-sensitive enzymatic activities. Nonetheless, all of the cal1/cdc43 mutants show temperature-sensitive growth phenotypes. Increase in soluble pools of the small GTPases was observed in the yeast mutant cells at the restrictive temperature in vivo, suggesting that the yeast prenylation pathway itself is temperature sensitive. The cal1-1 mutation, located most proximal to the C-terminus of the protein, differs from the other cdc43 mutations in several respects. An increase in soluble Rho1p was observed in the cal1-1 strain grown at the restrictive temperature. The temperature-sensitive phenotype of cal1-1 is most efficiently suppressed by overproduction of Rho1p. Overproduction of the other essential target, Cdc42p, in contrast, is deleterious in cal1-1 cells, but not in other cdc43 mutants or the wild-type strains. The cdc43-5 mutant cells accumulate Cdc42p in soluble pools and cdc43-5 is suppressed by overproduction of Cdc42p. Thus, several phenotypic differences are observed among the cal1/cdc43 mutations, possibly due to alterations in substrate specificity caused by the mutations.

Abstract

The Rho-type GTPase Cdc42p is required for cell polarization and bud emergence in Saccharomyces cerevisiae. To identify genes whose functions are linked to CDC42, we screened for (i) multicopy suppressors of a Ts- cdc42 mutant, (ii) mutants that require multiple copies of CDC42 for survival, and (iii) mutations that display synthetic lethality with a partial-loss-of-function allele of CDC24, which encodes a guanine nucleotide exchange factor for Cdc42p. In all three screens, we identified a new gene, BEM4. Cells from which BEM4 was deleted were inviable at 37 degrees C. These cells became unbudded, large, and round, consistent with a model in which Bem4p acts together with Cdc42p in polarity establishment and bud emergence. In some strains, the ability of CDC42 to serve as a multicopy suppressor of the Ts- growth defect of deltabem4 cells required co-overexpression of Rho1p, which is an essential Rho-type GTPase necessary for cell wall integrity. This finding suggests that Bem4p also affects Rho1p function. Bem4p displayed two-hybrid interactions with Cdc42p, Rho1p, and two of the three other known yeast Rho-type GTPases, suggesting that Bem4p can interact with multiple Rho-type GTPases. Models for the role of Bem4p include that it serves as a chaperone or modulates the interaction of these GTPases with one or more of their targets or regulators.

Abstract

Previous analysis of the bipolar budding pattern of Saccharomyces cerevisiae has suggested that it depends on persistent positional signals that mark the region of the division site and the tip of the distal pole on a newborn daughter cell, as well as each previous division site on a mother cell. In an attempt to identify genes encoding components of these signals or proteins involved in positioning or responding to them, we identified 11 mutants with defects in bipolar but not in axial budding. Five mutants displaying a bipolar budding-specific randomization of budding pattern had mutations in four previously known genes (BUD2, BUD5, SPA2, and BNI1) and one novel gene (BUD6), respectively. As Bud2p and Bud5p are known to be required for both the axial and bipolar budding patterns, the alleles identified here probably encode proteins that have lost their ability to interact with the bipolar positional signals but have retained their ability to interact with the distinct positional signal used in axial budding. The function of Spa2p is not known, but previous work has shown that its intracellular localization is similar to that postulated for the bipolar positional signals. BNI1 was originally identified on the basis of genetic interaction with CDC12, which encodes one of the neck-filament-associated septin proteins, suggesting that these proteins may be involved in positioning the bipolar signals. One mutant with a heterogeneous budding pattern defines a second novel gene (BUD7). Two mutants budding almost exclusively from the proximal pole carry mutations in a fourth novel gene (BUD9). A bud8 bud9 double mutant also buds almost exclusively from the proximal pole, suggesting that Bud9p is involved in positioning the proximal pole signal rather than being itself a component of this signal.

Abstract

The Saccharomyces cerevisiae CDC3, CDC10, CDC11, and CDC12 genes encode a family of related proteins, the septins, which are involved in cell division and the organization of the cell surface during vegetative growth. A search for additional S. cerevisiae septin genes using the polymerase chain reaction identified SPR3, a gene that had been identified previously on the basis of its sporulation-specific expression. The predicted SPR3 product shows 25-40% identity in amino acid sequence to the previously known septins from S. cerevisiae and other organisms. Immunoblots confirmed the sporulation-specific expression of Spr3p and showed that other septins are also present at substantial levels in sporulating cells. Consistent with the expression data, deletion of SPR3 in either of two genetic backgrounds had no detectable effect on exponentially growing cells. In one genetic background, deletion of SPR3 produced a threefold reduction in sporulation efficiency, although meiosis appeared to be completed normally. In this background, deletion of CDC10 had no detectable effect on sporulation. In the other genetic background tested, the consequences of the two deletions were reversed. Immunofluorescence observations suggest that Spr3p, Cdc3p, and Cdc11p are localized to the leading edges of the membrane sacs that form near the spindle-pole bodies and gradually extend to engulf the nuclear lobes that contain the haploid chromosome sets, thus forming the spores. Deletion of SPR3 does not prevent the localization of Cdc3p and Cdc11p, but these proteins appear to be less well organized, and the intensity of their staining is reduced. Taken together, the results suggest that the septins play important but partially redundant roles during the process of spore formation.

Abstract

The septins are a novel family of proteins that were first recognized in yeast as proteins associated with the neck filaments. Recent work has shown that septins are also present in other fungi, insects, and vertebrates. Despite the apparent differences in modes of cytokinesis amongst species, septins appear to be essential for this process in both fungal and animal cells. The septins also appear to be involved in various other aspects of the organization of the cell surface.

Abstract

We have selected yeast mutants that exhibit a constitutively active pheromone-response pathway in the absence of the beta subunit of the trimeric G protein. Genetic analysis of one such mutant revealed that it contained recessive mutations in two distinct genes, both of which contributed to the constitutive phenotype. One mutation identifies the RGA1 locus (Rho GTPase activating protein), which encodes a protein with homology to GAP domains and to LIM domains. Deletion of RGA1 is sufficient to activate the pathway in strains lacking the G beta subunit. Moreover, in wild-type strains, deletion of RGA1 increases signaling in the pheromone pathway, whereas over-expression of RGA1 dampens signaling, demonstrating that Rga1p functions as a negative regulator of the pheromone response pathway. The second mutation present in the original mutant proved to be an allele of a known gene, PBS2, which encodes a putative protein kinase that functions in the high osmolarity stress pathway. The pbs2 mutation enhanced the rga1 mutant phenotype, but by itself did not activate the pheromone pathway. Genetic and two-hybrid analyses indicate that an important target of Rga1p is Cdc42p, a p21 GTPase required for polarity establishment and bud emergence. This finding coupled with recent experiments with mammalian and yeast cells indicating that Cdc42p can interact with and activate Ste20p, a protein kinase that operates in the pheromone pathway, leads us to suggest that Rga1p controls the activity of Cdc42p, which in turn controls the magnitude of signaling in the pheromone pathway via Ste20p.

Abstract

The septins are a family of homologous proteins that were originally identified in Saccharomyces cerevisiae, where they are associated with the "neck filaments" and are involved in cytokinesis and other aspects of the organization of the cell surface. We report here the identification of Sep1, a Drosophila melanogaster septin, based on its homology to the yeast septins. The predicted Sep1 amino acid sequence is 35-42% identical to the known S. cerevisiae septins; 52% identical to Pnut, a second D. melanogaster septin; and 53-73% identical to the known mammalian septins. Sep1-specific antibodies have been used to characterize its expression and localization. The protein is concentrated at the leading edge of the cleavage furrows of dividing cells and cellularizing embryos, suggesting a role in furrow formation. Other aspects of Sep1 localization suggest roles not directly related to cytokinesis. For example, Sep1 exhibits orderly, cell-cycle-coordinated rearrangements within the cortex of syncytial blastoderm embryos and in the cells of post-gastrulation embryos; Sep1 is also concentrated at the leading edge of the epithelium during dorsal closure in the embryo, in the neurons of the embryonic nervous system, and at the baso-lateral surfaces of ovarian follicle cells. The distribution of Sep1 typically overlaps, but is distinct from, that of actin. Both immunolocalization and biochemical experiments show that Sep1 is intimately associated with Pnut, suggesting that the Drosophila septins, like those in yeast, function as part of a complex.

Abstract

In the budding yeast Saccharomyces cerevisiae, the process of conjugation of haploid cells of genotype MATa and MAT alpha to form MATa/alpha diploids is triggered by pheromones produced by each mating type. These pheromones stimulate a cellular response by interaction with receptors linked to a heterotrimeric G protein. Although genetic analysis indicates that the pheromone signal is transmitted through the G beta gamma dimer, the initial target(s) of G protein activation remain to be determined. Temperature-sensitive cells with mutations of the CDC24 and CDC42 genes, which are incapable of budding and of generating cell polarity at the restrictive temperature, are also unable to mate. Cdc24 acts as a guanylyl-nucleotide-exchange factor for the Rho-type GTPase Cdc42, which has been shown to be a fundamental component of the molecular machinery controlling morphogenesis in eukaryotic cells. Therefore, the inability of cdc24 and cdc42 mutants to mate has been presumed to be due to a requirement for generation of cell polarity and related morphogenetic events during conjugation. But here we show that Cdc42 has a direct signalling role in the mating-pheromone response between the G protein and the downstream protein kinase cascade.

Abstract

The yeast Ste20 protein kinase is involved in pheromone response. Mammalian homologs of Ste20 exist, but their function remains unknown. We identified a novel yeast STE20 homolog, CLA4, in a screen for mutations lethal in the absence of the G1 cyclins Cln1 and Cln2. Cla4 is involved in budding and cytokinesis and interacts with Cdc42, a GTPase required for polarized cell growth. Despite a cytokinesis defect, cla4 mutants are viable. However, double cla4 ste20 mutants cannot maintain septin rings at the bud neck and cannot undergo cytokinesis. Mutations in CDC12, which encodes one of the septins, were found in the same screen. Cla4 and Ste20 kinases apparently share a function in localizing cell growth with respect to the septin ring.

Abstract

Yeast cells can select bud sites in either of two distinct spatial patterns. a cells and alpha cells typically bud in an axial pattern, in which both mother and daughter cells form new buds adjacent to the preceding division site. In contrast, a/alpha cells typically bud in a bipolar pattern, in which new buds can form at either pole of the cell. The BUD3 gene is specifically required for the axial pattern of budding: mutations of BUD3 (including a deletion) affect the axial pattern but not the bipolar pattern. The sequence of BUD3 predicts a product (Bud3p) of 1635 amino acids with no strong or instructive similarities to previously known proteins. However, immunofluorescence localization of Bud3p has revealed that it assembles in an apparent double ring encircling the mother-bud neck shortly after the mitotic spindle forms. The Bud3p structure at the neck persists until cytokinesis, when it splits to yield a single ring of Bud3p marking the division site on each of the two progeny cells. These single rings remain for much of the ensuing unbudded phase and then disassemble. The Bud3p rings are indistinguishable from those of the neck filament-associated proteins (Cdc3p, Cdc10p, Cdc11p, and Cdc12p), except that the latter proteins assemble before bud emergence and remain in place for the duration of the cell cycle. Upon shift of a temperature-sensitive cdc12 mutant to restrictive temperature, localization of both Bud3p and the neck filament-associated proteins is rapidly lost. In addition, a haploid cdc11 mutant loses its axial-budding pattern upon shift to restrictive temperature. Taken together, the data suggest that Bud3p and the neck filaments are linked in a cycle in which each controls the position of the other's assembly: Bud3p assembles onto the neck filaments in one cell cycle to mark the site for axial budding (including assembly of the new ring of neck filaments) in the next cell cycle. As the expression and localization of Bud3p are similar in a, alpha, and a/alpha cells, additional regulation must exist such that Bud3p restricts the position of bud formation in a and alpha cells but not in a/alpha cells.

Abstract

Cells of the yeast Saccharomyces cerevisiae select bud sites in either of two distinct spatial patterns, known as axial (expressed by a and alpha cells) and bipolar (expressed by a/alpha cells). Fluorescence, time-lapse, and scanning electron microscopy have been used to obtain more precise descriptions of these patterns. From these descriptions, we conclude that in the axial pattern, the new bud forms directly adjacent to the division site in daughter cells and directly adjacent to the immediately preceding division site (bud site) in mother cells, with little influence from earlier sites. Thus, the division site appears to be marked by a spatial signal(s) that specifies the location of the new bud site and is transient in that it only lasts from one budding event to the next. Consistent with this conclusion, starvation and refeeding of axially budding cells results in the formation of new buds at nonaxial sites. In contrast, in bipolar budding cells, both poles are specified persistently as potential bud sites, as shown by the observations that a pole remains competent for budding even after several generations of nonuse and that the poles continue to be used for budding after starvation and refeeding. It appears that the specification of the two poles as potential bud sites occurs before a daughter cell forms its first bud, as a daughter can form this bud near either pole. However, there is a bias towards use of the pole distal to the division site. The strength of this bias varies from strain to strain, is affected by growth conditions, and diminishes in successive cell cycles. The first bud that forms near the distal pole appears to form at the very tip of the cell, whereas the first bud that forms near the pole proximal to the original division site (as marked by the birth scar) is generally somewhat offset from the tip and adjacent to (or overlapping) the birth scar. Subsequent buds can form near either pole and appear almost always to be adjacent either to the birth scar or to a previous bud site. These observations suggest that the distal tip of the cell and each division site carry persistent signals that can direct the selection of a bud site in any subsequent cell cycle.

Abstract

Forty-eight mutants of Saccharomyces cerevisiae with defects in glycogen metabolism were isolated. The mutations defined eight GLC genes, the function of which were determined. Mutations in three of these genes activate the RAS/cAMP pathway either by impairment of a RAS GTPase-activating protein (GLC1/IRA1 and GLC4/IRA2) or by activating Ras2p (GLC5/RAS2). SNF1 protein kinase (GLC2) was found to be required for normal glycogen levels. Glycogen branching enzyme (GLC3) was found to be required for significant glycogen synthesis. GLC6 was shown to be allelic to CIF1 (and probably FDP1, BYP1 and GGS1), mutations in which were previously found to prevent growth on glucose; this gene is also the same as TPS1, which encodes a subunit of the trehalose-phosphate synthase. Mutations in GLC6 were capable of increasing or decreasing glycogen levels, at least in part via effects on the regulation of glycogen synthase. GLC7 encodes a type 1 protein phosphatase that contributes to the dephosphorylation (and hence activation) of glycogen synthase. GLC8 encodes a homologue of type 1 protein phosphatase inhibitor-2. The genetic map positions of GLC1/IRA1, GLC3, GLC4/IRA2, GLC6/CIF1/TPS1 (and the adjacent VAT2/VMA2), and GLC7 were clarified. From the data on GLC3, there may be a suppression of recombination near the chromosome V centromere, at least in some strains.

Abstract

Geranylgeranyl transferase I (GGTase I), which modifies proteins containing the sequence Cys-Ali-Ali-Leu (Ali: aliphatic) at their C-termini, is indispensable for growth in the budding yeast Saccharomyces cerevisiae. We report here that GGTase I is no longer essential when Rho1p and Cdc42p are simultaneously overproduced. The lethality of a GGTase I deletion is most efficiently suppressed by provision of both Rho1p and Cdc42p with altered C-terminal sequences (Cys-Ali-Ali-Met) corresponding to the C-termini of substrates of farnesyl transferase (FTase). Under these circumstances, the FTase, normally not essential for growth of yeast, becomes essential.

Abstract

Previous analyses of Saccharomyces cerevisiae chromosome I have suggested that the majority (greater than 75%) of single-copy essential genes on this chromosome are difficult or impossible to identify using temperature-sensitive (Ts-) lethal mutations. To investigate whether this situation reflects intrinsic difficulties in generating temperature-sensitive proteins or constraints on mutagenesis in yeast, we subjected three cloned essential genes from chromosome I to mutagenesis in an Escherichia coli mutator strain and screened for Ts- lethal mutations in yeast using the "plasmid-shuffle" technique. We failed to obtain Ts- lethal mutations in two of the genes (FUN12 and FUN20), while the third gene yielded such mutations, but only at a low frequency. DNA sequence analysis of these mutant alleles and of the corresponding wild-type region revealed that each mutation was a single substitution not in the previously identified gene FUN19, but in the adjacent, newly identified essential gene FUN53. FUN19 itself proved to be non-essential. These results suggest that many essential proteins encoded by genes on chromosome I cannot be rendered thermolabile by single mutations. However, the results obtained with FUN53 suggest that there may also be significant constraints on mutagenesis in yeast. The 5046 base-pair interval sequenced contains the complete FUN19, FUN53 and FUN20 coding regions, as well as a portion of the adjacent non-essential FUN21 coding region. In all, 68 to 75% of this interval is open reading frame. None of the four predicted products shows significant homologies to known proteins in the available databases.

Abstract

MSB2 was identified previously as a multicopy suppressor of a temperature-sensitive mutation in CDC24, a gene required for polarity establishment and bud formation in Saccharomyces cerevisiae. The inferred MSB2 product contains 1306 amino acids, 42% of which are Ser or Thr. Its Ser+Thr-richness and hydrophobicity profile suggest that Msb2p may be an integral membrane protein containing a long, periplasmic, N-terminal domain and a short, cytoplasmic, C-terminal domain. Cells that lack MSB2 display no obvious mutant phenotypes. MSB2 is located between the centromere and KSS1 on the right arm of chromosome VII. Although physical mapping suggests that MSB2 and LEU1 (on the left arm of chromosome VII) are approximately 40 kb apart, the genetic map distance observed between leu1 and an msb2::URA3 marker was only 2.3 cM.

Abstract

Cell polarization requires that a cellular axis or cell-surface site be chosen and that the cytoskeleton be organized with respect to it. Details of the link between the cytoskeleton and the chosen axis or site are not clear. Cells of the yeast Saccharomyces cerevisiae exhibit cell polarization in two phases of their life cycle, during vegetative growth and during mating, which reflects responses to intracellular and extracellular signals, respectively. Here we describe the isolation of two mutants defective specifically in cell polarization in response to peptide mating pheromones. The mutants carry special alleles (denoted bem1-s) of the BEM1 gene required for cell polarization during vegetative growth. Unlike other bem1 mutants, the bem1-s mutants are normal for vegetative growth. Complete deletion of BEM1 leads to the defect in polarization of vegetative cells seen in bem1 mutants. The predicted sequence of the BEM1 protein (Bem1p) reveals two copies of a domain (denoted SH3) that is found in many proteins associated with the cortical cytoskeleton and which may mediate binding to actin or some other component of the cell cortex. The sequence of Bem1p and the properties of mutants defective in this protein indicate that it may link the cytoskeleton to morphogenetic determinants on the cell surface.

Abstract

The Saccharomyces cerevisiae ras-like gene RSR1 is particularly closely related to the mammalian gene Krev-1 (also known as smg21A and rap1A). RSR1 was originally isolated as a multicopy suppressor of a cdc24 mutation, which causes an inability to bud or establish cell polarity. Deletion of RSR1 itself does not affect growth but causes a randomization of bud position. We have now constructed mutant alleles of RSR1 encoding proteins with substitutions of Val for Gly at position 12 (analogous to constitutively activated Ras proteins) or Asn for Lys at position 16 (analogous to a dominant-negative Ras protein). rsr1Val-12 could not restore a normal budding pattern to an rsr1 deletion strain but could suppress a cdc24 mutation when overexpressed. rsr1Asn-16 could randomize the budding pattern of a wild-type strain even in low copy number but was not lethal even in high copy number. These and other results suggest that Rsr1p functions only in bud site selection and not in subsequent events of polarity establishment and bud formation, that this function involves a cycling between GTP-bound and GDP-bound forms of the protein, and that the suppression of cdc24 involves direct interaction between Rsr1p[GTP] and Cdc24p. Functional homology between Rsr1p and Krev-1 p21 was suggested by the observations that expression of the latter protein in yeast cells could both suppress a cdc24 mutation and randomize the budding pattern of wild-type cells. As Krev-1 overexpression can suppress ras-induced transformation of mammalian cells, we looked for effects of RSR1 on the S. cerevisiae Ras pathway. Although no suppression of the activated RAS2Val-19 allele was observed, overexpression of rsr1Val-12 suppressed the lethality of strains lacking RAS gene function, apparently through a direct activation of adenyl cyclase. This interaction of Rsr1p with the effector of Ras in S. cerevisiae suggests that Krev-1 may revert ras-induced transformation of mammalian cells by affecting the interaction of ras p21 with its effector.

Abstract

Microscopic screening of a collection of cold-sensitive mutants of Saccharomyces cerevisiae led to the identification of a new gene, CDC55, which appears to be involved in the morphogenetic events of the cell cycle. CDC55 maps between CDC43 and CHC1 on the left arm of chromosome VII. At restrictive temperature, the original cdc55 mutant produces abnormally elongated buds and displays a delay or partial block of septation and/or cell separation. A cdc55 deletion mutant displays a cold-sensitive phenotype like that of the original isolate. Sequencing of CDC55 revealed that it encodes a protein of about 60 kDa, as confirmed by Western immunoblots using Cdc55p-specific antibodies. This protein has greater than 50% sequence identity to the B subunits of rabbit skeletal muscle type 2A protein phosphatase; the latter sequences were obtained by analysis of peptides derived from the purified protein, a polymerase chain reaction product, and cDNA clones. An extragenic suppressor of the cdc55 mutation lies in BEM2, a gene previously identified on the basis of an apparent role in bud emergence.

Abstract

Budding by yeast follows a sequence of three stages. These include selection of a non-random bud-site, organization of that site and establishment of an associated axis of cytoskeletal polarity, and localized growth of the cell surface to produce the bud. Numerous components involved in each stage have been identified. As some of these components have close homologs in other organisms, there may exist common mechanisms involved in the establishment of cell polarity.

Abstract

Cells of the yeast S. cerevisiae choose bud sites in an axial or bipolar spatial pattern depending on their cell type. We have identified a gene, BUD5, that resembles BUD1 and BUD2 in being required for both patterns; bud5- mutants also exhibit random budding in all cell types. The BUD5 nucleotide sequence predicts a protein of 538 amino acids that has similarity to the S. cerevisiae CDC25 product, an activator of RAS proteins that catalyzes GDP-GTP exchange. Two potential targets of BUD5 are known: BUD1 (RSR1) and CDC42, proteins involved in bud site selection and bud formation, respectively, that have extensive similarity to RAS. We also show that BUD5 interacts functionally with a gene, BEM1, that is required for bud formation. This interaction provides further support for the view that products involved in bud site selection guide the positioning of a complex necessary for bud formation.

USE OF A SCREEN FOR SYNTHETIC LETHAL AND MULTICOPY SUPPRESSEE MUTANTS TO IDENTIFY 2 NEW GENES INVOLVED IN MORPHOGENESIS IN SACCHAROMYCES-CEREVISIAEMOLECULAR AND CELLULAR BIOLOGYBender, A., Pringle, J. R.1991; 11 (3): 1295-1305

Abstract

Genes CDC24 and CDC42 are required for the establishment of cell polarity and for bud formation in Saccharomyces cerevisiae. Temperature-sensitive (Ts-) mutations in either of these genes cause arrest as large, unbudded cells in which the nuclear cycle continues. MSB1 was identified previously as a multicopy suppressor of Ts- cdc24 and cdc42 mutations. We have now sequenced MSB1 and constructed a deletion of this gene. The predicted amino acid sequence does not closely resemble any other in the available data bases, and the deletion does not produce any readily detectable phenotype. However, we have used a colony-sectoring assay to identify additional genes that appear to interact with MSB1 and play a role in bud emergence. Starting with a strain deleted for the chromosomal copy of MSB1 but containing MSB1 on a high-copy-number plasmid, mutants were identified in which MSB1 had become essential for viability. The new mutations defined two genes, BEM1 and BEM2; both the bem1 and bem2 mutations are temperature sensitive and are only partially suppressed by MSB1. In bem1 cells, a single copy of MSB1 is necessary and sufficient for viability at 23 or 30 degrees C, but even multiple copies of MSB1 do not fully suppress the growth defect at 37 degrees C. In bem2 cells, a single copy of MSB1 is necessary and sufficient for viability at 23 degrees C, multiple copies are necessary for viability at 30 degrees C, and even multiple copies of MSB1 do not suppress the growth defect at 37 degrees C. In a wild-type background (i.e., a single chromosomal copy of MSB1), both bem1 and bem2 mutations cause cells to become large and multinucleate even during growth at 23 degrees C, suggesting that these genes are involved in bud emergence. This suggestion is supported for BEM1 by other evidence obtained in a parallel study (J. Chant, K. Corrado, J. Pringle, and I. Herskowitz, submitted for publication). BEM1 maps centromere distal to TYR1 on chromosome II, and BEM2 maps between SPT15 and STP2 on chromosome V.

GENETIC-ANALYSIS OF SACCHAROMYCES-CEREVISIAE CHROMOSOME-I - ON THE ROLE OF MUTAGEN SPECIFICITY IN DELIMITING THE SET OF GENES IDENTIFIABLE USING TEMPERATURE-SENSITIVE-LETHAL MUTATIONSGENETICSHarris, S. D., Pringle, J. R.1991; 127 (2): 279-285

Abstract

In a previous attempt to identify as many as possible of the essential genes on Saccharomyces cerevisiae chromosome I, temperature-sensitive (Ts-) lethal mutations that had been induced by ethyl methane-sulfonate or nitrosoguanidine were analyzed. Thirty-two independently isolated mutations that mapped to chromosome I identified only three complementation groups, all of which had been known previously. In contrast, molecular analyses of segments of the chromosome have suggested the presence of numerous additional essential genes. In order to assess the degree to which problems of mutagen specificity had limited the set of genes detected using Ts- lethal mutations, we isolated a new set of such mutations after mutagenesis with UV or nitrogen mustard. Surprisingly, of 21 independently isolated mutations that mapped to chromosome I, 17 were again in the same three complementation groups as identified previously, and two of the remaining four mutations were apparently in a known gene involved in cysteine biosynthesis. Of the remaining two mutations, one was in one of the essential genes identified in the molecular analyses, and the other was too leaky to be mapped. These results suggest that only a minority of the essential genes in yeast can be identified using Ts- lethal mutations, regardless of the mutagen used, and thus emphasize the need to use multiple genetic strategies in the investigation of cellular processes.

Abstract

Saccharomyces cerevisiae chromosome I has provided a vivid example of the "gene-number paradox." Although molecular studies have suggested that there are greater than 100 transcribed regions on the chromosome, classical genetic studies have identified only about 15 genes, including just 6 identified in intensive studies using Ts- lethal mutations. To help elucidate the reasons for this disparity, we have undertaken a detailed molecular analysis of a 34-kb segment of the left arm of the chromosome. This segment contains the four known genes CDC24, WHI1, CYC3 and PYK1 plus at least seven transcribed regions of unknown function. The 11 identified transcripts have a total length of approximately 25.9 kb, suggesting that greater than or equal to 75% of the DNA in this region is transcribed. Of the transcribed regions of unknown function, three are essential for viability on rich medium and three appear to be nonessential, as judged by the lethality or nonlethality of deletions constructed using integrative transformation methods. No obvious phenotypes were associated with the deletions in the apparently nonessential genes. However, two of these genes may have homologs elsewhere in the genome, as judged from the appearance of additional bands when DNA-DNA blot hybridizations were performed at reduced stringency. Taken together, the results provide further evidence that the limitations of classical genetic studies of chromosome I cannot be explained solely by a lack of genes, or even a lack of essential genes, on the chromosome.

Abstract

Budding cells of the yeast Saccharomyces cerevisiae possess a ring of 10-nm-diameter filaments, of unknown biochemical nature, that lies just inside the plasma membrane in the neck connecting the mother cell to its bud. Electron microscopic observations suggest that these filaments assemble at the budding site coincident with bud emergence and disassemble shortly before cytokinesis (Byers, B. and L. Goetsch. 1976. J. Cell Biol. 69:717-721). Mutants defective in any of four genes (CDC3, CDC10, CDC11, or CDC12) lack these filaments and display a pleiotropic phenotype that involves abnormal bud growth and an inability to complete cytokinesis. We showed previously by immunofluorescence that the CDC12 gene product is probably a constituent of the ring of 10-nm filaments (Haarer, B. and J. Pringle. 1987. Mol. Cell. Biol. 7:3678-3687). We now report the use of fusion proteins to generate polyclonal antibodies specific for the CDC3 gene product. In immunofluorescence experiments, these antibodies decorated the neck regions of wild-type and mutant cells in patterns suggesting that the CDC3 gene product is also a constituent of the ring of 10-nm filaments. We also used the CDC3-specific and CDC12-specific antibodies to investigate the timing of localization of these proteins to the budding site. The results suggest that the CDC3 protein is organized into a ring at the budding site well before bud emergence and remains so organized for some time after cytokinesis. The CDC12 product appears to behave similarly, but may arrive at the budding site closer to the time of bud emergence, and disappear from that site more quickly after cytokinesis, than does the CDC3 product. Examination of mating cells and cells responding to purified mating pheromone revealed novel arrangements of the CDC3 and CDC12 products in the regions of cell wall reorganization. Both proteins were present in normal-looking ring structures at the bases of the first zygotic buds.

CELLULAR MORPHOGENESIS IN THE SACCHAROMYCES-CEREVISIAE CELL-CYCLE - LOCALIZATION OF THE CDC11 GENE-PRODUCT AND THE TIMING OF EVENTS AT THE BUDDING SITEDEVELOPMENTAL GENETICSFord, S. K., Pringle, J. R.1991; 12 (4): 281-292

Abstract

The Saccharomyces cerevisiae CDC3, CDC10, CDC11, and CDC12 genes encode a family of homologous proteins that are not closely related to other known proteins [Haarer BK, Ketcham SR, Ford SK, Ashcroft DJ, and Pringle JR (submitted)]. Temperature-sensitive mutants defective in any of these four genes display essentially identical pleiotropic phenotypes that include abnormal cell-wall deposition and bud growth, an inability to complete cytokinesis, and a failure to form the ring of 10 nm filaments that normally lies directly subjacent to the plasma membrane in the neck region of budding cells. We showed previously that the CDC3 and CDC12 gene products localize to the region of the mother-bud neck and are probably constituents of the ring of 10 nm filaments. We now report the generation of polyclonal antibodies specific for the CDC11 product (Cdc11p) and the use of these antibodies in immunofluorescence experiments with wild-type and mutant cells. The results suggest that Cdc11p is also a constituent of the filament ring, and thus support the hypothesis that the S. cerevisiae 10 nm filaments represent a novel type of eukaryotic cytoskeletal element. Cdc11p and actin both localize to the budding site well in advance of bud emergence and at approximately the same time, and both proteins also remain localized at the old budding site for some time after cytokinesis. Cdc11p also localizes to regions of cell-wall reorganization in mating cells and in cells responding to purified mating pheromone. Surprisingly, most preparations of affinity purified Cdc11p-specific antibodies also stained the nuclear and cytoplasmic microtubules. Although this staining probably reflects the existence of an epitope shared by Cdc11p and some microtubule-associated protein, the possibility that a fraction of the Cdc11p is associated with the microtubules could not be eliminated.

Abstract

Budding in the yeast Saccharomyces cerevisiae involves a polarized deposition of new cell surface material that is associated with a highly asymmetric disposition of the actin cytoskeleton. Mutants defective in gene CDC24, which are unable to bud or establish cell polarity, have been of great interest with regard to both the mechanisms of cellular morphogenesis and the mechanisms that coordinate cell-cycle events. To gain further insights into these problems, we sought additional mutants with defects in budding. We report here that temperature-sensitive mutants defective in genes CDC42 and CDC43, like cdc24 mutants, fail to bud but continue growth at restrictive temperature, and thus arrest as large unbudded cells. Nearly all of the arrested cells appear to begin nuclear cycles (as judged by the occurrence of DNA replication and the formation and elongation of mitotic spindles), and many go on to complete nuclear division, supporting the hypothesis that the events associated with budding and those of the nuclear cycle represent two independent pathways within the cell cycle. The arrested mutant cells display delocalized cell-surface deposition associated with a loss of asymmetry of the actin cytoskeleton. CDC42 maps distal to the rDNA on chromosome XII and CDC43 maps near lys5 on chromosome VII.

Abstract

The Saccharomyces cerevisiae CDC42 gene product is involved in the morphogenetic events of the cell division cycle; temperature-sensitive cdc42 mutants are unable to form buds and display delocalized cell-surface deposition at the restrictive temperature (Adams, A. E. M., D. I. Johnson, R. M. Longnecker, B. F. Sloat, and J. R. Pringle. 1990. J. Cell Biol. 111:131-142). To begin a molecular analysis of CDC42 function, we have isolated the CDC42 gene from a yeast genomic DNA library. The use of the cloned DNA to create a deletion of CDC42 confirmed that the gene is essential. Overexpression of CDC42 under control of the GAL10 promoter was not grossly deleterious to cell growth but did perturb the normal pattern of selection of budding sites. Determination of the DNA and predicted amino acid sequences of CDC42 revealed a high degree of similarity in amino acid sequence to the ras and rho (Madaule, P., R. Axel, and A. M. Myers. 1987. Proc. Natl. Acad. Sci. 84:779-783) families of gene products. The similarities to ras proteins (approximately 40% identical or related amino acids overall) were most pronounced in the regions that have been implicated in GTP binding and hydrolysis and in the COOH-terminal modifications leading to membrane association, suggesting that CDC42 function also involves these biochemical properties. The similarities to the rho proteins (approximately 60% identical or related amino acids overall) were more widely distributed through the coding region, suggesting more extensive similarities in as yet undefined biochemical properties and functions.

MULTICOPY SUPPRESSION OF THE CDC24 BUDDING DEFECT IN YEAST BY CDC42 AND 3 NEWLY IDENTIFIED GENES INCLUDING THE RAS-RELATED GENE RSR1PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICABender, A., Pringle, J. R.1989; 86 (24): 9976-9980

Abstract

Genes CDC24, CDC42, and CDC43 are required for the establishment of cell polarity and the localization of secretion in Saccharomyces cerevisiae; mutants defective in these genes fail to form buds and display isotropic expansion of the cell surface. To identify other genes that may be involved in these processes, we screened yeast genomic DNA libraries for heterologous genes that, when overexpressed from a plasmid, can suppress a temperature-sensitive cdc24 mutation. We identified four such genes. One of these proved to be CDC42, which has previously been shown to be a member of the rho (ras-homologous) family of genes, and a second is a newly identified ras-related gene that we named RSR1. RSR1 maps between CDC62 and ADE3 on the right arm of chromosome VII; its predicted product is approximately 50% identical to other proteins in the ras family. Deletion of RSR1 is nonlethal but disrupts the normal pattern of bud site selection. Although both CDC42 and RSR1 can suppress cdc24 and both appear to encode GTP-binding proteins, these genes do not themselves appear to be functionally interchangeable. However, one of the other genes that was isolated by virtue of its ability to suppress cdc24 can also suppress cdc42. This gene, named MSB1, maps between ADE9 and HIS3 on the right arm of chromosome XV.

Abstract

We used the inhibitor nocodazole in conjunction with immunofluorescence and electron microscopy to investigate microtubule function in the yeast cell cycle. Under appropriate conditions, this drug produced a rapid and essentially complete disassembly of cytoplasmic and intranuclear microtubules, accompanied by a rapid and essentially complete block of cellular and nuclear division. These effects were similar to, but more profound than, the effects of the related drug methyl benzimidazole carbamate (MBC). In the nocodazole-treated cells, the selection of nonrandom budding sites, the formation of chitin rings and rings of 10-nm filaments at those sites, bud emergence, differential bud enlargement, and apical bud growth appeared to proceed normally, and the intracellular distribution of actin was not detectably perturbed. Thus, the cytoplasmic microtubules are apparently not essential for the establishment of cell polarity and the localization of cell-surface growth. In contrast, nocodazole profoundly affected the behavior of the nucleus. Although spindle-pole bodies (SPBs) could duplicate in the absence of microtubules, SPB separation was blocked. Moreover, complete spindles present at the beginning of drug treatment appeared to collapse, drawing the opposed SPBs and associated nuclear envelope close together. Nuclei did not migrate to the mother-bud necks in nocodazole-treated cells, although nuclei that had reached the necks before drug treatment remained there. Moreover, the double SPBs in arrested cells were often not oriented toward the budding sites, in contrast to the situation in normal cells. Thus, microtubules (cytoplasmic, intranuclear, or both) appear to be necessary for the migration and proper orientation of the nucleus, as well as for SPB separation, spindle function, and nuclear division.

Abstract

CDC3, CDC25 and CDC42 were localized to chromosome XII by hybridizing the cloned genes to Southern blots of chromosomes separated by orthogonal-field-alternation gel electrophoresis. Meiotic tetrad analyses further localized these genes to the region distal to the RDN1 locus on the right arm of the chromosome. The STE11 gene, which had previously been mapped to chromosome XII (Chaleff and Tatchell, 1985), was found to be tightly linked to ILV5. The data suggest a map order of CEN12-RDN1-CDC42-(CDC25-CDC3)-(ILV5- STE11)-URA4. Certain oddities of the data set raise the possibility that there may be constraints on the patterns of recombination in this region of chromosome XII.

IMMUNOFLUORESCENCE LOCALIZATION OF THE SACCHAROMYCES-CEREVISIAE CDC12 GENE-PRODUCT TO THE VICINITY OF THE 10-NM FILAMENTS IN THE MOTHER-BUD NECKMOLECULAR AND CELLULAR BIOLOGYHaarer, B. K., Pringle, J. R.1987; 7 (10): 3678-3687

Abstract

Budding cells of the yeast Saccharomyces cerevisiae possess a ring of 10-nm-diameter filaments, of unknown biochemical nature, that lies just inside the plasma membrane in the neck connecting the mother cell to its bud (B. Byers and L. Goetsch, J. Cell Biol. 69:717-721, 1976). Mutants defective in any of four genes (CDC3, CDC10, CDC11, and CDC12) lack these filaments and display a pleiotropic phenotype that involves abnormal bud growth and cell-wall deposition and an inability to complete cytokinesis. We fused the cloned CDC12 gene to the Escherichia coli lacZ and trpE genes and used the resulting fusion proteins to raise polyclonal antibodies specific for the CDC12 gene product. In immunofluorescence experiments with affinity-purified antibodies, the neck region of wild-type and mutant cells stained in patterns consistent with the hypothesis that the CDC12 gene product is a constituent of the ring of 10-nm filaments. Without careful affinity purification of the CDC12-specific antibodies, these staining patterns were completely obscured by the staining of residual cell wall components in the neck by antibodies present even in the "preimmune" sera of all rabbits tested.

Abstract

Molecular cloning techniques were used to isolate and characterize the DNA including and surrounding the CDC24 and PYK1 genes on the left arm of chromosome I of the yeast Saccharomyces cerevisiae. A plasmid that complemented a temperature-sensitive cdc24 mutation was isolated from a yeast genomic DNA library in a shuttle vector. Plasmids containing pyk1-complementing DNA were obtained from other investigators. Several lines of evidence (including one-step gene replacement experiments) demonstrated that the complementing plasmids contained the bona fide CDC24 and PYK1 genes. These sequences were then used to isolate additional DNA from chromosome I by probing a yeast genomic DNA library in a lambda vector. A total of 28 kilobases (kb) of contiguous DNA surrounding the CDC24 and PYK1 genes was isolated, and a restriction map was determined. Electron microscopy of R-loop-containing DNA and RNA blot hybridization analyses indicated that an 18-kb segment contained at least seven transcribed regions, only three of which corresponded to previously known genes (CDC24, PYK1, and CYC3). Southern blot hybridization experiments suggested that none of the genes in this region was duplicated elsewhere in the yeast genome. The centers of CDC24 and PYK1 were only approximately 7.5 kb apart, although the genetic map distance between them is approximately 13 centimorgans. As previous studies with S. cerevisiae have indicated that 1 centimorgan generally corresponds to approximately 3 kb, the region between CDC24 and PYK1 appears to undergo meiotic recombination at an unusually high frequency.

Abstract

The distribution of actin in wild-type cells and in morphogenetic mutants of the budding yeast Saccharomyces cerevisiae was explored by staining cells with fluorochrome-labeled phallotoxins after fixing and permeabilizing the cells by several methods. The actin appeared to be localized in a set of cortical spots or patches, as well as in a network of cytoplasmic fibers. Bundles of filaments that may possibly correspond to the fibers visualized by fluorescence were observed with the electron microscope. The putative actin spots were concentrated in small and medium-sized buds and at what were apparently the sites of incipient bud formation on unbudded cells, whereas the putative actin fibers were generally oriented along the long axes of the mother-bud pairs. In several morphogenetic mutants that form multiple, abnormally elongated buds, the actin patches were conspicuously clustered at the tips of most buds, and actin fibers were clearly oriented along the long axes of the buds. There was a strong correlation between the occurrence of active growth at particular bud tips and clustering of actin spots at those same tips. Near the end of the cell cycle in wild-type cells, actin appeared to concentrate (as a cluster of spots or a band) in the neck region connecting the mother cell to its bud. Observations made using indirect immunofluorescence with a monoclonal anti-yeast-tubulin antibody on the morphogenetic mutant cdc4 (which forms multiple, abnormally elongated buds while the nuclear cycle is arrested) revealed the surprising occurrence of multiple bundles of cytoplasmic microtubules emanating from the one duplicated spindle-pole body per cell. It seems that most or all of the buds contain one or more of these bundles of microtubules, which often can be seen to extend to the very tips of the buds. These observations are consistent with the hypotheses that actin, tubulin, or both may be involved in the polarization of growth and localization of cell-wall deposition that occurs during the yeast cell cycle.

Abstract

A method was developed for isolating large numbers of mutations on chromosome I of the yeast Saccharomyces cerevisiae. A strain monosomic for chromosome I (i.e., haploid for chromosome I and diploid for all other chromosomes) was mutagenized with either ethyl methanesulfonate or N-methyl-N'-nitro-N-nitrosoguanidine and screened for temperature-sensitive (Ts-) mutants capable of growth on rich, glucose-containing medium at 25 degrees but not at 37 degrees. Recessive mutations induced on chromosome I are expressed whereas those on the diploid chromosomes are usually not expressed because of the presence of wild-type alleles on the homologous chromosomes. Dominant ts mutations on all chromosomes should also be expressed, but these appeared rarely.--Of the 41 ts mutations analyzed, 32 mapped on chromosome I. These 32 mutations fell into only three complementation groups, which proved to be the previously described genes CDC15, CDC24 and PYK1 (or CDC19). We recovered 16 or 17 independent mutations in CDC15, 12 independent mutations in CDC24 and three independent mutations in PYK1. A fourth gene on chromosome I, MAK16, is known to be capable of giving rise to a ts-lethal allele, but we recovered no mutations in this gene. The remaining nine mutations isolated using the monosomic strain appeared not to map on chromosome I and were apparently expressed in the original mutants because they had become homozygous or hemizygous by mitotic recombination or chromosome loss.--The available information about the size of chromosome I suggests that it should contain approximately 60-100 genes. However, our isolation in the monosomic strain of multiple, independent alleles of just three genes suggests that only a small proportion of the genes on chromosome I is easily mutable to give a Ts--lethal phenotype.--During these studies, we located CDC24 on chromosome I and determined that it is centromere distal to PYK1 on the left arm of the chromosome.

Abstract

Exponentially growing populations were abruptly shifted to media lacking a nitrogen source, a sulphur source or a phosphorus source. When proliferation ceased, cells were homogeneously arrested at the beginning of the cell cycle and were resistant to killing by exposure to 52 degrees C and to cell wall degrading enzymes. The results suggest that these two types of resistance represent a general response to nutrient limitation and are characteristic of resting cells.

Abstract

Temperature-sensitive yeast mutants defective in gene CDC24 continued to grow (i.e., increase in cell mass and cell volume) at restrictive temperature (36 degrees C) but were unable to form buds. Staining with the fluorescent dye Calcofluor showed that the mutants were also unable to form normal bud scars (the discrete chitin rings formed in the cell wall at budding sites) at 36 degrees C; instead, large amounts of chitin were deposited randomly over the surfaces of the growing unbudded cells. Labeling of cell-wall mannan with fluorescein isothiocyanate-conjugated concanavalin A suggested that mannan incorporation was also delocalized in mutant cells grown at 36 degrees C. Although the mutants have well-defined execution points just before bud emergence, inactivation of the CDC24 gene product in budded cells led both to selective growth of mother cells rather than of buds and to delocalized chitin deposition, indicating that the CDC24 gene product functions in the normal localization of growth in budded as well as in unbudded cells. Growth of the mutant strains at temperatures less than 36 degrees C revealed allele-specific differences in behavior. Two strains produced buds of abnormal shape during growth at 33 degrees C. Moreover, these same strains displayed abnormal localization of budding sites when growth at 24 degrees C (the normal permissive temperature for the mutants); in each case, the abnormal pattern of budding sites segregated with the temperature sensitivity in crosses. Thus, the CDC24 gene product seems to be involved in selection of the budding site, formation of the chitin ring at that site, the subsequent localization of new cell wall growth to the budding site and the growing bud, and the balance between tip growth and uniform growth of the bud that leads to the normal cell shape.

Abstract

We have identified a yeast protein that resembles actins from other eucaryotes in its tight binding to pancreatic deoxyribonuclease I, its copolymerizaton with purified muscle actin, its one-dimensional peptide map, and its apparent polymerization into 7-nm filaments. The yeast actin-like protein yielded a single spot on two-dimensional polyacrylamide gel electrophoresis, suggesting that a single protein species was present. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the actin-like protein had an apparent molecular weight of 45,000 compared with 42,000 for muscle actin. In an attempt to identify the messenger ribonucleic acid coding for the actin-like protein, yeast polyadenylic acid-rich ribonucleic acid was translated in wheat germ and reticulocyte cell-free protein-synthesizing systems. The actin-like protein was identified among the translation products of the reticulocyte system by its tight binding to deoxyribonuclease I, its comigration with the in vivo-synthesized actin-like protein during sodium dodecyl sulfate-polyacrylamide gel electrophoresis, an the similarity of its peptide map to that of the in vivo-synthesized protein. A yeast protein synthesized in the wheat-germ system was also found to bind to deoxyribonuclease I and to copolymerize with muscle actin. However, its apparent molecular weight was about 35,000, suggesting that it was a product either of incomplete translation or of proteolytic cleavage of the actin-like protein.

Abstract

The resistance of exponentially growing yeast cells to killing by exposure to 52 degrees C increase markedly as the growth temperature was increased. Identical killing curves were obtained for cells suspended in growth medium or in 0.9% saline. Cells resistant to killing at 52 degrees C were quite sensitive to killing at slightly higher temperatures. These results suggest a primary role for membrane damage in the mechanism of heat killing.

Abstract

The amounts of glycogen and trehalose have been measured in cells of a prototrophic diploid yeast strain subjected to a variety of nutrient limitations. Both glycogen and trehalose were accumulated in cells deprived specifically of nirogen, sulfur, or phosphorus, suggesting that reserve carbohydrate accumulation is a general response to nutrient limitation. The patterns of accumulation and utilization of glycogen and trehalose were not identical under these conditions, suggesting that the two carbohydrates may play distinct physiological roles. Glycogen and trehalose were also accumulated by cells undergoing carbon and energy limitation, both during diauxic growth in a relatively poor medium and during the approach to stationary phase in a rich medium. Growth in the rich medium was shown to be carbon or energy limited or both, although the interaction between carbon source limitation and oxygen limitation was complex. In both media, the pattern of glycogen accumulation and utilization was compatible with its serving as a source of energy both during respiratory adaptation and during a subsequent starvation. In contrast, the pattern of trehalose accumulation and utilization seemed compatible only with the latter role. In cultures that were depleting their supplies of exogenous glucose, the accumulation of glycogen began at glucose concentrations well above those sufficient to suppress glycogen accumulation in cultures growing with a constant concentration of exogenous glucose. The mechanism of this effect is not clear, but may involve a response to the rapid rate of change in the glucose concentration.

Abstract

In the budding yeast Saccharomyces cerevisiae, each bud appears within a ring of chitin formed in the cell wall of the mother cell. Temperature-sensitive mutants defective in gene cdc24 synthesize chitin at restrictive temperatures, but do not organize it into the discrete rings found in normal cells, nor do they form buds. The chitin ring or an annular precursor structure may play an essential role in reinforcing the region of the cell wall involved in budding.